Soluble recombinant botulinum toxin proteins

ABSTRACT

The present invention includes recombinant proteins derived from  Clostridium botulinum  toxins. In particular, soluble recombinant  Clostridium botulinum  type A, type B and type E toxin proteins are provided. Methods which allow for the isolation of recombinant proteins free of significant endotoxin contamination are provided. The soluble, endotoxin-free recombinant proteins are used as immunogens for the production of vaccines and antitoxins. These vaccines and antitoxins are useful in the treatment of humans and other animals at risk of intoxication with clostridial toxin.

[0001] This application is a Continuation-In-Part of copendingapplication Ser. No. 08/405,496, filed Mar. 16, 1995.

FIELD OF THE INVENTION

[0002] The present invention relates to the isolation of polypeptidesderived from Clostridium botulinum neurotoxins and the use thereof asimmunogens for the production of vaccines, including multivalentvaccines, and antitoxins.

BACKGROUND OF THE INVENTION

[0003] The genus Clostridium is comprised of gram-positive, anaerobic,spore-forming bacilli. The natural habitat of these organisms is theenvironment and the intestinal tracts of humans and other animals.Indeed, clostridia are ubiquitous; they are commonly found in soil,dust, sewage, marine sediments, decaying vegetation, and mud. [See e.g.,P. H. A. Sneath et al., “Clostridium,” Bergey's Manual® of SystematicBacteriology, Vol. 2, pp. 1141-1200, Williams & Wilkins (1986). Despitethe identification of approximately 100 species of Clostridium, only asmall number have been recognized as etiologic agents of medical andveterinary importance. Nonetheless, these species are associated withvery serious diseases, including botulism, tetanus, anaerobiccellulitis, gas gangrene, bacteremia, pseudomembranous colitis, andclostridial gastroenteritis. Table 1 lists some of the species ofmedical and veterinary importance and the diseases with which they areassociated. As virtually all of these species have been isolated fromfecal samples of apparently healthy persons, some of these isolates maybe transient, rather than permanent residents of the colonic flora.TABLE 1 Clostridium Species Of Medical And Veterinary Importance*Species Disease C. aminovalericum Bacteriuria (pregnant women) C.argentinense Infected wounds; Bacteremia; Botulism; Infections ofamniotic fluid C. baratii Infected war wounds; Peritonitis; Infectiousprocesses of the eye, ear and prostate C. beijerinckikii Infected woundsC. bifermentans Infected wounds; Abscesses; Gas Gangrene; Bacteremia C.botulinum Food poisoning; Botulism (wound, food, infant) C. butyricumUrinary tract, lower respiratory tract, pleural cavity, and abdominalinfections; Infected wounds; Abscesses; Bacteremia C. cadaverisAbscesses; Infected wounds C. carnis Soft tissue infections; BacteremiaC. chauvoei Blackleg C. clostridioforme Abdominal, cervical, scrotal,pleural, and other infections; Septicemia; Peritonitis; Appendicitis C.cochlearium Isolated from human disease processes, but role in diseaseunknown. C. difficile Antimicrobial-associated diarrhea;Pseudomembranous enterocolitis; Bacteremia; Pyogenic infections C.fallax Soft tissue infections C. ghnoii Soft tissue infections C.glycolicum Wound infections; Abscesses; Peritonitis C. hastiformeInfected war wounds; Bacteremia; Abscesses C. histolyticum Infected warwounds; Gas gangrene; Gingival plaque isolate C. indolisGastrointestinal tract infections C. innocuum Gastrointestinal tractinfections; Empyema C. irregulare Penile lesions C. leptum Isolated fromhuman disease processes, but role in disease unknown. C. limosumBacteremia; Peritonitis; Pulmonary infections C. malenominatum Variousinfectious processes C. novyi Infected wounds; Gas gangrene; Blackleg,Big head (ovine); Redwater disease (bovine) C. oroticum Urinary tractinfections; Rectal abscesses C. paraputrificum Bacteremia; Peritonitis;Infected wounds; Appendicitis C. perfringens Gas gangrene; Anaerobiccellulitis; Intra-abdominal abscesses; Soft tissue infections; Foodpoisoning; Necrotizing pneumonia; Empyema; Meningitis; Bacteremia;Uterine Infections; Enteritis necrotans; Lamb dysentery; Struck; OvineEnterotoxemia; C. putrefaciens Bacteriuria (Pregnant women withbacteremia) C. putrificum Abscesses; Infected wounds; Bacteremia C.ramosum Infections of the abdominal cavity, genital tract, lung, andbiliary tract; Bacteremia C. sartagoforme Isolated from human diseaseprocesses, but role in disease unknown. C. septicum Gas gangrene;Bacteremia; Suppurative infections; Necrotizing enterocolitis; Braxy C.sordellii Gas gangrene; Wound infections; Penile lesions; Bacteremia;Abscesses; Abdominal and vaginal infections C. sphenoides Appendicitis;Bacteremia; Bone and soft tissue infections; Intraperitoneal infections;Infected war wounds; Visceral gas gangrene; Renal abscesses C.sporogenes Gas gangrene; Bacteremia; Endocarditis; central nervoussystem and pleuropulmonary infections; Penile lesions; Infected warwounds; Other pyogenic infections C. subterminale Bacteremia; Empyema;Biliary tract, soft tissue and bone infections C. symbiosum Liverabscesses; Bacteremia; Infections resulting due to bowel flora C.tertium Gas gangrene; Appendicitis; Brain abscesses; Intestinal tractand soft tissue infections; Infected war wounds; Periodontitis;Bacteremia C. tetani Tetanus; Infected gums and teeth; Cornealulcerations; Mastoid and middle ear infections; Intraperitonealinfections; Tetanus neonatorum; Postpartum uterine infections; Softtissue infections, especially related to trauma (including abrasions andlacerations); Infections related to use of contaminated needles C.thermosaccharolyticum Isolated from human disease processes, but role indisease unknown. # Toxins, 2d ed., pp. 66-67, American Society forMicrobiology (1986); R. Berkow and A. J. Fletcher (eds.), “BacterialDiseases,” Merck Manual of Diagnosis and Therapy, 16th ed., pp. 116-126,Merck Research Laboratories, Rahway, N. J. (1992); and O. H. Sigmund andC. M. Fraser (eds.), # “Clostridial Infections,”Merck Veterinary Manual,5th ed., pp. 396-409, Merck & Co., Rahway, N. J. (1979).

[0004] In most cases, the pathogenicity of these organisms is related tothe release of powerful exotoxins or highly destructive enzymes. Indeed,several species of the genus Clostridium produce toxins and otherenzymes of great medical and veterinary significance. [C. L. Hatheway,Clin. Microbiol. Rev. 3:66-98 (1990).]

[0005] Perhaps because of their significance for human and veterinarymedicine, much research has been conducted on these toxins, inparticular those of C. botulinum and C. difficile.

[0006]C. botulinum

[0007] Several strains of Clostridium botulinum produce toxins ofsignificance to human and animal health. [C. L. Hatheway, Clin.Microbiol. Rev. 3:66-98 (1990)] The effects of these toxins range fromdiarrheal diseases that can cause destruction of the colon, to paralyticeffects that can cause death. Particularly at risk for developingclostridial diseases are neonates and humans and animals in poor health(e.g., those suffering from diseases associated with old age orimmunodeficiency diseases).

[0008]Clostridium botulinum produces the most poisonous biological toxinknown. The lethal human dose is a mere 10⁻⁹ mg/kg bodyweight for toxinin the bloodstream. Botulinal toxin blocks nerve transmission to themuscles, resulting in flaccid paralysis. When the toxin reaches airwayand respiratory muscles, it results in respiratory failure that cancause death. [S. Arnon, J. Infect. Dis. 154:201-206 (1986)]

[0009]C. botulinum spores are carried by dust and are found onvegetables taken from the soil, on fresh fruits, and on agriculturalproducts such as honey. Under conditions favorable to the organism, thespores germinate to vegetative cells which produces toxin. [S. Amon,Ann. Rev. Med. 31:541 (1980)]

[0010] Botulism disease may be grouped into four types, based on themethod of introduction of toxin into the bloodstream. Food-bornebotulism results from ingesting improperly preserved and inadequatelyheated food that contains botulinal toxin. There were 355 cases offood-borne botulism in the United States between 1976 and 1984. [K. L.MacDonald et al., Am. J. Epidemiol. 124:794 (1986).] The death rate dueto botulinal toxin is 12% and can be higher in particular risk groups.[C. O. Tacket et al., Am. J. Med. 76:794 (1984).] Wound-induced botulismresults from C. botulinum penetrating traumatized tissue and producingtoxin that is absorbed into the bloodstream. Since 1950, thirty cases ofwound botulism have been reported. [M. N. Swartz, “AnaerobicSpore-Forming Bacilli: The Clostridia,” pp. 633-646, in B. D. Davis etal.,(eds.), Microbiology, 4th edition, J. B. Lippincott Co. (1990).]Inhalation botulism results when the toxin is inhaled. Inhalationbotulism has been reported as the result of accidental exposure in thelaboratory [E. Holzer, Med. Klin. 41:1735 (1962)] and could arise if thetoxin is used as an agent of biological warfare [D. R. Franz et al., inBotulinum and Tetanus Neurotoxins, B. R. DasGupta, ed., Plenum Press,New York (1993), pp. 473-476]. Infectious infant botulism results fromC. botulinum colonization of the infant intestine with production oftoxin and its absorption into the bloodstream. It is likely that thebacterium gains entry when spores are ingested and subsequentlygerminate. [S. Amon, J. Infect. Dis. 154:201 (1986).] There have been500 cases reported since it was first recognized in 1976. [M. N. Swartz,supra.]

[0011] Infant botulism strikes infants who are three weeks to elevenmonths old (greater than 90% of the cases are infants less than sixmonths). [S. Arnon, J. Infect. Dis. 154:201 (1986).] It is believed thatinfants are susceptible, due, in large part, to the absence of the fulladult complement of intestinal microflora. The benign microflora presentin the adult intestine provide an acidic environment that is notfavorable to colonization by C. botulinum. Infants begin life with asterile intestine which is gradually colonized by microflora. Because ofthe limited microflora present in early infancy, the intestinalenvironment is not as acidic, allowing for C. botulinum sporegermination, growth, and toxin production. In this regard, some adultswho have undergone antibiotic therapy which alters intestinal microflorabecome more susceptible to botulism.

[0012] An additional factor accounting for infant susceptibility toinfectious botulism is the immaturity of the infant immune system. Themature immune system is sensitized to bacterial antigens and producesprotective antibodies. Secretory IgA produced in the adult intestine hasthe ability to agglutinate vegetative cells of C. botulinum. [S. Arnon,J. Infect. Dis. 154:201 (1986).] Secretory IgA may also act bypreventing intestinal bacteria and their products from crossing thecells of the intestine. [S. Amon, Epidemiol. Rev. 3:45 (1981).] Theinfant immune system is not primed to do this.

[0013] Clinical symptoms of infant botulism range from mild paralysis,to moderate and severe paralysis requiring hospitalization, to fulminantparalysis, leading to sudden death. [S. Arnon, Epidemiol. Rev. 3:45(1981).]

[0014] The chief therapy for severe infant botulism is ventilatoryassistance using a mechanical respirator and concurrent elimination oftoxin and bacteria using cathartics, enemas, and gastric lavage. Therewere 68 hospitalizations in California for infant botulism in a singleyear with a total cost of over $4 million for treatment. [T. L.Frankovich and S. Arnon, West. J. Med. 154:103 (1991).]

[0015] Different strains of Clostridium botulinum each produceantigenically distinct toxin designated by the letters A-G. Serotype Atoxin has been implicated in 26% of the cases of food botulism; types B,E and F have also been implicated in a smaller percentage of the foodbotulism cases [H. Sugiyama, Microbiol. Rev. 44:419 (1980)]. Woundbotulism has been reportedly caused by only types A or B toxins [H.Sugiyama, supra]. Nearly all cases of infant botulism have been causedby bacteria producing either type A or type B toxin. (Exceptionally, oneNew Mexico case was caused by Clostridium botulinum producing type Ftoxin and another by Clostridium botulinum producing a type B-type Fhybrid.) [S. Amon, Epidemiol. Rev. 3:45 (1981).] Type C toxin affectswaterfowl, cattle, horses and mink. Type D toxin affects cattle, andtype E toxin affects both humans and birds.

[0016] A trivalent antitoxin derived from horse plasma is commerciallyavailable from Connaught Industries Ltd. as a therapy for toxin types A,B, and E. However, the antitoxin has several disadvantages. First,extremely large dosages must be injected intravenously and/orintramuscularly. Second, the antitoxin has serious side effects such asacute anaphylaxis which can lead to death, and serum sickness. Finally,the efficacy of the antitoxin is uncertain and the treatment is costly.[C. O. Tacket et al., Am. J. Med. 76:794 (1984).]

[0017] A heptavalent equine botulinal antitoxin which uses only theF(ab′)2 portion of the antibody molecule has been tested by the UnitedStates Military. [M. Balady, USAMRDC Newsletter, p. 6 (1991).] This wasraised against impure toxoids in those large animals and is not a hightiter preparation.

[0018] A pentavalent human antitoxin has been collected from immunizedhuman subjects for use as a treatment for infant botulism. The supply ofthis antitoxin is limited and cannot be expected to meet the needs ofall individuals stricken with botulism disease. In addition, collectionof human sera must involve screening out HIV and other potentiallyserious human pathogens. [P. J. Schwarz and S. S. Amon, Western J. Med.156:197 (1992).]

[0019] Infant botulism has been implicated as the cause of mortality insome cases of Sudden Infant Death Syndrome (SIDS, also known as cribdeath). SIDS is officially recognized as infant death that is sudden andunexpected and that remained unexplained despite complete post-mortemexamination. The link of SIDS to infant botulism came when fecal orblood specimens taken at autopsy from SIDS infants were found to containC. botulinum organisms and/or toxin in 3-4% of cases analyzed. [D. R.Peterson et al., Rev. Infect. Dis. 1:630 (1979).] In contrast, only 1 of160 healthy infants (0.6%) had C. botulinum organisms in the feces andno botulinal toxin. (S. Amon et al., Lancet, pp. 1273-76, Jun. 17,1978.)

[0020] In developed countries, SIDS is the number one cause of death inchildren between one month and one year old. (S. Arnon et al., Lancet,pp. 1273-77, Jun. 17, 1978.) More children die from SIDS in the firstyear than from any other single cause of death in the first fourteenyears of life. In the United States, there are 8,000-10,000 SIDS victimsannually. Id.

[0021] What is needed is an effective therapy against infant botulismthat is free of dangerous side effects, is available in large supply ata reasonable price, and can be safely and gently delivered so thatprophylactic application to infants is feasible.

[0022] Immunization of subjects with toxin preparations has been done inan attempt to induce immunity against botulinal toxins. A C. botulinumvaccine comprising chemically inactivated (i.e., formaldehyde-treated)type A, B, C, D and E toxin is commercially available for human usage.However, this vaccine preparation has several disadvantages. First, theefficacy of this vaccine is variable (in particular, only 78% ofrecipients produce protective levels of anti-type B antibodies followingadministration of the primary series). Second, immunization is painful(deep subcutaneous inoculation is required for administration), withadverse reactions being common (moderate to severe local reactions occurin approximately 6% of recipients upon initial injection; this numberrises to approximately 11% of individuals who receive boosterinjections) [Informational Brochure for the Pentavalent (ABCDE)Botulinum Toxoid, Centers for Disease Control]. Third, preparation ofthe vaccine is dangerous as active toxin must be handled by laboratoryworkers.

[0023] What is needed are safe and effective vaccine preparations foradministration to those at risk of exposure to C. botulinum toxins.

[0024]C. difficile

[0025]C. difficile, an organism which gained its name due todifficulties encountered in its isolation, has recently been proven tobe an etiologic agent of diarrheal disease. (Sneath et al., p. 1165.).C. difficile is present in the gastrointestinal tract of approximately3% of healthy adults, and 10-30% of neonates without adverse effect(Swartz, at p. 644); by other estimates, C. difficile is a part of thenormal gastrointestinal flora of 2-10% of humans. [G. F. Brooks et al.,(eds.) “Infections Caused by Anaerobic Bacteria,” Jawetz, Melnick, &Adelberg's Medical Microbiology, 19th ed., pp. 257-262, Appleton &Lange, San Mateo, Calif. (1991).] As these organisms are relativelyresistant to most commonly used antimicrobials, when a patient istreated with antibiotics, the other members of the normalgastrointestinal flora are suppressed and C. difficile flourishes,producing cytopathic toxins and enterotoxins. It has been found in 25%of cases of moderate diarrhea resulting from treatment with antibiotics,especially the cephalosporins, clindamycin, and ampicillin. [M. N.Swartz at 644.]

[0026] Importantly, C. difficile is commonly associated with nosocomialinfections. The organism is often present in the hospital and nursinghome environments and may be carried on the hands and clothing ofhospital personnel who care for debilitated and immunocompromisedpatients. As many of these patients are being treated withantimicrobials or other chemotherapeutic agents, such transmission of C.difficile represents a significant risk factor for disease. (Engelkirket al., pp. 64-67.) C. difficile is associated with a range ofdiarrhetic illness, ranging from diarrhea alone to marked diarrhea andnecrosis of the gastrointestinal mucosa with the accumulation ofinflammatory cells and fibrin, which forms a pseudomembrane in theaffected area. (Brooks et al.) It has been found in over 95% ofpseudomembranous enterocolitis cases. (Swartz, at p. 644.) Thisoccasionally fatal disease is characterized by diarrhea, multiple smallcolonic plaques, and toxic megacolon. (Swartz, at p. 644.) Althoughstool cultures are sometimes used for diagnosis, diagnosis is best madeby detection of the heat labile toxins present in fecal filtrates frompatients with enterocolitis due to C. difficile. (Swartz, at p. 644-645;and Brooks et al., at p. 260.) C. difficile toxins are cytotoxic fortissue/cell cultures and cause enterocolitis when injected intracecallyinto hamsters. (Swartz, at p. 644.)

[0027] The enterotoxicity of C. difficile is primarily due to the actionof two toxins, designated A and B, each of approximately 300,000 inmolecular weight. Both are potent cytotoxins, with toxin A possessingdirect enterocytotoxic activity. [Lyerly et al., Infect. Immun. 60:4633(1992).] Unlike toxin A of C. perfringens, an organism rarely associatedwith antimicrobial-associated diarrhea, the toxin of C. difficile is nota spore coat constituent and is not produced during sporulation.(Swartz, at p. 644.) C. difficile toxin A causes hemorrhage, fluidaccumulation and mucosal damage in rabbit ileal loops and appears toincrease the uptake of toxin B by the intestinal mucosa. Toxin B doesnot cause intestinal fluid accumulation, but it is 1000 times more toxicthan toxin A to tissue culture cells and causes membrane damage.Although both toxins induce similar cellular effects such as actindisaggregation, differences in cell specificity occurs.

[0028] Both toxins are important in disease. [Borriello et al., Rev.Infect. Dis., 12(suppl. 2):S185 (1990); Lyerly et al., Infect. Immun.,47:349 (1985); and Rolfe, Infect. Immun., 59:1223 (1990).] Toxin A isthought to act first by binding to brush border receptors, destroyingthe outer mucosal layer, then allowing toxin B to gain access to theunderlying tissue. These steps in pathogenesis would indicate that theproduction of neutralizing antibodies against toxin A may be sufficientin the prophylactic therapy of CDAD. However, antibodies against toxin Bmay be a necessary additional component for an effective therapeuticagainst later stage colonic disease. Indeed, it has been reported thatanimals require antibodies to both toxin A and toxin B to be completelyprotected against the disease. [Kim and Rolfe, Abstr. Ann. Meet. Am.Soc. Microbiol., 69:62 (1987).]

[0029]C. difficile has also been reported to produce other toxins suchas an enterotoxin different from toxins A and B [Banno et at, Rev.Infect. Dis., 6(Suppl. 1:S11-S20 (1984)], a low molecular weight toxin[Rihn et al., Biochem. Biophys. Res. Comm., 124:690-695 (1984)], amotility altering factor [Justus et al., Gastroenterol., 83:836-843(1982)], and perhaps other toxins. Regardless, C. difficilegastrointestinal disease is of primary concern.

[0030] It is significant that due to its resistance to most commonlyused antimicrobials, C. difficile is associated with antimicrobialtherapy with virtually all antimicrobial agents (although most commonlyampicillin, clindamycin and cephalosporins). It is also associated withdisease in patients undergoing chemotherapy with such compounds asmethotrexate, 5-fluorouracil, cyclophosphamide, and doxorubicin. [S. M.Finegold et al., Clinical Guide to Anaerobic Infections, pp. 88-89, StarPublishing Co., Belmont, Calif. (1992).]

[0031] Treatment of C. difficile disease is problematic, given the highresistance of the organism. Oral metronidazole, bacitracin andvancomycin have been reported to be effective. (Finegold et al., p. 89.)However there are problems associated with treatment utilizing thesecompounds. Vancomycin is very expensive, some patients are unable totake oral medication, and the relapse rate is high (20-25%), although itmay not occur for several weeks. Id.

[0032]C. difficile disease would be prevented or treated by neutralizingthe effects of these toxins in the gastrointestinal tract. Thus, what isneeded is an effective therapy against C. difficile toxin that is freeof dangerous side effects, is available in large supply at a reasonableprice, and can be safely delivered so that prophylactic application topatients at risk of developing pseudomembranous enterocolitis can beeffectively treated.

DESCRIPTION OF THE DRAWINGS

[0033]FIG. 1 shows the reactivity of anti-C. botulinum IgY by Westernblot.

[0034]FIG. 2 shows the IgY antibody titer to C, botulinum type A toxoidin eggs, measured by ELISA.

[0035]FIG. 3 shows the results of C. difficile toxin A neutralizationassays.

[0036]FIG. 4 shows the results of C. difficile toxin B neutralizationassays.

[0037]FIG. 5 shows the results of C. difficile toxin B neutralizationassays.

[0038]FIG. 6 is a restriction map of C. difficile toxin A gene, showingsequences of primers 1-4 (SEQ ID NOS:1-4).

[0039]FIG. 7 is a Western blot of C. difficile toxin A reactive protein.

[0040]FIG. 8 shows C. difficile toxin A expression constructs.

[0041]FIG. 9 shows C. difficile toxin A expression constructs.

[0042]FIG. 10 shows the purification of recombinant C. difficile toxinA.

[0043]FIG. 11 shows the results of C. difficile toxin A neutralizationassays with antibodies reactive to recombinant toxin A.

[0044]FIG. 12 shows the results for a C. difficile toxin Aneutralization plate.

[0045]FIG. 13 shows the results for a C. difficile toxin Aneutralization plate.

[0046]FIG. 14 shows the results of recombinant C. difficile toxin Aneutralization assays.

[0047]FIG. 15 shows C. difficile toxin A expression constructs.

[0048]FIG. 16 shows a chromatograph plotting absorbance at 280 nmagainst retention time for a pMA1870-680 IgY PEG preparation.

[0049]FIG. 17 shows two recombinant C. difficile toxin B expressionconstructs.

[0050]FIG. 18 shows C. difficile toxin B expression constructs.

[0051]FIG. 19 shows C. difficile toxin B expression constructs.

[0052]FIG. 20 shows C. difficile toxin B expression constructs.

[0053]FIG. 21 is an SDS-PAGE gel showing the purification of recombinantC. difficile toxin B fusion protein.

[0054]FIG. 22 is an SDS-PAGE gel showing the purification of twohistidine-tagged recombinant C. difficile toxin B proteins.

[0055]FIG. 23 shows C. difficile toxin B expression constructs.

[0056]FIG. 24 is a Western blot of C. difficile toxin B reactiveprotein.

[0057]FIG. 25 shows C. botulinum type A toxin expression constructs;constructs used to provide C. botulinum or C. difficile sequences arealso shown.

[0058]FIG. 26 is an SDS-PAGE gel stained with Coomaisse blue showing thepurification of recombinant C. botulinum type A toxin fusion proteins.

[0059]FIG. 27 shows C. botulinum type A toxin expression constructs;constructs used to provide C. botulinum sequences are also shown.

[0060]FIG. 28 is an SDS-PAGE gel stained with Coomaisse blue showing thepurification of pHisBot protein using the Ni-NTA resin.

[0061]FIG. 29 is an SDS-PAGE gel stained with Coomaisse blue showing theexpression of pHisBot protein in BL21(DE3) and BL21(DE3)pLysS hostcells.

[0062]FIG. 30 is an SDS-PAGE gel stained with Coomaisse blue showing thepurification of pHisBot protein using a batch absorption procedure.

[0063]FIG. 31 is an SDS-PAGE gel stained with Coomaisse blue showing thepurification of pHisBot and pHisBot(native) proteins using a Ni-NTAcolumn.

[0064]FIG. 32 is an SDS-PAGE gel stained with Coomaisse blue showing thepurification of pHisBotA protein expressed in pHisBotA(syn) kan lacIqT7/pACYCGro/BL21(DE3) cells using an IDA column.

[0065]FIG. 33 is an SDS-PAGE gel stained with Coomaisse blue showing thepurification of pHisBotA, pHisBotB and pHisBotE proteins by IDAchromatography followed by chromatography on S-100 to remove foldingchaperones.

[0066]FIG. 34 is an SDS-PAGE gel stained with Coomaisse blue showing theextracts derived from pHisBotB amp T7lac/BL21(DE3) cells before andafter purification on a Ni-NTA column.

[0067]FIG. 35 is an SDS-PAGE gel run under native conditions and stainedwith Coomaisse blue showing the removal of folding chaperones fromIDA-purified BotB protein using a S-100 column.

[0068]FIG. 36 is an SDS-PAGE gel stained with Coomaisse blue showingproteins that eluted during an imidazole step gradient applied to a IDAcolumn containing a lysate of pHisBotB kan lacIq T7/pACYCGro/BL21(DE3)cells.

[0069]FIG. 37 is an SDS-PAGE gel run under native conditions and stainedwith Coomaisse blue showing IDA-purified BotB protein before and afterultrafiltration.

[0070]FIG. 38 is an SDS-PAGE gel stained with Coomaisse blue showing thepurification of BotE protein using a NiNTA column.

[0071]FIG. 39 is an SDS-PAGE gel stained with Coomaisse blue showingextracts derived from pHisBotA kan T7 lac/BL21(DE3) pLysS cells grown infermentation culture.

[0072]FIG. 40 is a chromatogram showing proteins present afterIDA-purified BotE protein was applied to a S-100 column.

[0073] Definitions

[0074] To facilitate understanding of the invention, a number of termsare defined below.

[0075] As used herein, the term “neutralizing” is used in reference toantitoxins, particularly antitoxins comprising antibodies, which havethe ability to prevent the pathological actions of the toxin againstwhich the antitoxin is directed.

[0076] As used herein, the term “overproducing” is used in reference tothe production of clostridial toxin polypeptides in a host cell andindicates that the host cell is producing more of the clostridial toxinby virtue of the introduction of nucleic acid sequences encoding saidclostridial toxin polypeptide than would be expressed by said host cellabsent the introduction of said nucleic acid sequences. To allow ease ofpurification of toxin polypeptides produced in a host cell it ispreferred that the host cell express or overproduce said toxinpolypeptide at a level greater than 1 mg/liter of host cell culture.

[0077] “A host cell capable of expressing a recombinant protein at alevel greater than or equal to 5% of the total cellular protein” is ahost cell in which the recombinant protein represents at least 5% of thetotal cellular protein. To determine what percentage of total cellularprotein the recombinant protein represents, the following steps aretaken. A total of 10 OD₆₀₀ units of recombinant host cells (e.g., 200 μlof cells at OD₆₀₀=50/ml) are removed (at a timepoint known to representthe peak of expression of the desired recombinant protein) to a 1.5 mlmicrofuge tube and pelleted for 2 min at maximum rpm in a microfuge. Thepellets are resuspended in 1 ml of 50 mM NaHPO₄, 0.5 M NaCl, 40 mMimidazole buffer (pH 6.8) containing 1 mg/ml lysozyme. The samples areincubated for 20 min at room temperature and stored ON at −70° C.Samples are thawed completely at room temperature and sonicated 2×10seconds with a Branson Sonifier 450 microtip probe at #3 power setting.The samples are centrifuged for 5 min. at maximum rpm in a microfuge. Analiquot (20 μl) of the protein sample is removed to 20 μl 2× samplebuffer (this represents the total protein extract). The samples areheated to 95° C. for 5 min, then cooled and 5 or 10 μl are loaded onto12.5% SDS-PAGE gels. High molecular weight protein markers are alsoloaded to allow for estimation of the MW of identified recombinantproteins. After electrophoresis, protein is detected generally bystaining with Coomassie blue and the stained gel is scanned using adensitometer to determine the percentage of protein present in eachband. In this manner, the percentage of protein present in the bandcorresponding to the recombinant protein of interest may be determined.It is not necessary that Coomassie blue be employed for the detection ofprotein, a number of fluorescent dyes [e.g., Sypro orange S-6651(Molecular Probes, Eugene, Oreg.] may be employed and the stained gelscanned using a fluoroimager [e.g., Fluor Imager SI (Molecular Dynamics,Sunnyvale, Calif.)].

[0078] “A host cell capable of expressing a recombinant protein as asoluble protein at a level greater than or equal to 0.25% of the totalsoluble cellular protein” is a host cell in which the amount of solublerecombinant protein present represents at least 0.25% of the totalcellular protein. As used herein “total soluble cellular protein” refersto a clarified PEI lysate prepared as described in Example 31(c)(iv).Briefly, cells are harvested following induction of expression ofrecombinant protein (at a point of maximal expression). The cells areresuspended in cell resuspension buffer (CRB: 50 mM NaPO₄, 0.5 M NaCl,40 mM imidazole, pH 6.8) to create a 20% cell suspension (wet weight ofcells/volume of CRB) and cell lysates are prepared as described inExample 31(c)(iv) (i.e., sonication or homogenization followed bycentrifugation). The cell lysate is then flocculated utilizingpolyethyleneimine (PEI) prior to centrifugation. PEI (a 2% solution indH₂O, pH 7.5 with HCl) is added to the cell lysate to a finalconcentration of 0.2%, and stirred for 20 min at room temperature priorto centrifugation [8,500 rpm in JAIO rotor (Beckman) for 30 minutes at4° C.]. This treatment removes RNA, DNA and cell wall components,resulting in a clarified, low viscosity lysate (“PEI clarified lysate”).The recombinant protein present in the PEI clarified lysate is thenpurified (e.g., by chromatography on an IDA column for his-taggedproteins). The amount of purified recombinant protein (i.e., the elutedprotein) is divided by the concentration of protein present in the PEIclarified lysate (typically 8 mg/ml when using a 20% cell suspension asthe starting material) and multiplied by 100 to determine whatpercentage of total soluble cellular protein is comprised of the solublerecombinant protein (see Example 33b).

[0079] As used herein, the term “fusion protein” refers to a chimericprotein containing the protein of interest (i.e., C. botulinum toxin A,B, C, D, E, F, or G and fragments thereof) joined to an exogenousprotein fragment (the fusion partner which consists of a non-toxinprotein). The fusion partner may enhance solubility of the C. botulinumprotein as expressed in a host cell, may provide an affinity tag toallow purification of the recombinant fusion protein from the host cellor culture supernatant, or both. If desired, the fusion protein may beremoved from the protein of interest (i.e., toxin protein or fragmentsthereof) prior to immunization by a variety of enzymatic or chemicalmeans known to the art.

[0080] As used herein the term “non-toxin protein” or “non-toxin proteinsequence” refers to that portion of a fusion protein which comprises aprotein or protein sequence which is not derived from a bacterial toxinprotein.

[0081] The term “protein of interest” as used herein refers to theprotein whose expression is desired within the fusion protein. In afusion protein the protein of interest will be joined or fused withanother protein or protein domain, the fusion partner, to allow forenhanced stability of the protein of interest and/or ease ofpurification of the fusion protein.

[0082] As used herein, the term “maltose binding protein” refers to themaltose binding protein of E. coli. A portion of the maltose bindingprotein may be added to a protein of interest to generate a fusionprotein; a portion of the maltose binding protein may merely enhance thesolubility of the resulting fusion protein when expressed in a bacterialhost. On the other hand, a portion of the maltose binding protein mayallow affinity purification of the fusion protein on an amylose resin.

[0083] As used herein, the term “poly-histidine tract” when used inreference to a fusion protein refers to the presence of two to tenhistidine residues at either the amino- or carboxy-terminus of a proteinof interest. A poly-histidine tract of six to ten residues is preferred.The poly-histidine tract is also defined functionally as being a numberof consecutive histidine residues added to the protein of interest whichallows the affinity purification of the resulting fusion protein on anickel-chelate or IDA column.

[0084] As used herein, the term “purified” or “to purify” refers to theremoval of contaminants from a sample. For example, antitoxins arepurified by removal of contaminating non-immunoglobulin proteins; theyare also purified by the removal of immunoglobulin that does not bindtoxin. The removal of non-immunoglobulin proteins and/or the removal ofimmunoglobulins that do not bind toxin results in an increase in thepercent of toxin-reactive immunoglobulins in the sample. In anotherexample, recombinant toxin polypeptides are expressed in bacterial hostcells and the toxin polypeptides are purified by the removal of hostcell proteins; the percent of recombinant toxin polypeptides is therebyincreased in the sample. Additionally, the recombinant toxinpolypeptides are purified by the removal of host cell components such aslipopolysaccharide (e.g., endotoxin).

[0085] The term “recombinant DNA molecule” as used herein refers to aDNA molecule which is comprised of segments of DNA joined together bymeans of molecular biological techniques.

[0086] The term “recombinant protein” or “recombinant polypeptide” asused herein refers to a protein molecule which is expressed from arecombinant DNA molecule.

[0087] The term “native protein” as used herein refers to a proteinwhich is isolated from a natural source as opposed to the production ofa protein by recombinant means.

[0088] As used herein the term “portion” when in reference to a protein(as in “a portion of a given protein”) refers to fragments of thatprotein. The fragments may range in size from four amino acid residuesto the entire amino acid sequence minus one amino acid.

[0089] As used herein “soluble” when in reference to a protein producedby recombinant DNA technology in a host cell is a protein which existsin solution in the cytoplasm of the host cell; if the protein contains asignal sequence the soluble protein is exported to the periplasmic spacein bacteria hosts and is secreted into the culture medium in eucaryoticcells capable of secretion or by bacterial host possessing theappropriate genes (i.e., the kil gene). In contrast, an insolubleprotein is one which exists in denatured form inside cytoplasmicgranules (called inclusion bodies) in the host cell. High levelexpression (i.e., greater than 10-20 mg recombinant protein/liter ofbacterial culture) of recombinant proteins often results in theexpressed protein being found in inclusion bodies in the bacterial hostcells. A soluble protein is a protein which is not found in an inclusionbody inside the host cell or is found both in the cytoplasm and ininclusion bodies and in this case the protein may be present at high orlow levels in the cytoplasm.

[0090] A distinction is drawn between a soluble protein (i.e., a proteinwhich when expressed in a host cell is produced in a soluble form) and a“solubilized” protein. An insoluble recombinant protein found inside aninclusion body may be solubilized (i.e., rendered into a soluble form)by treating purified inclusion bodies with denaturants such as guanidinehydrochloride, urea or sodium dodecyl sulfate (SDS). These denaturantsmust then be removed from the solubilized protein preparation to allowthe recovered protein to renature (refold). Not all proteins will refoldinto an active conformation after solubilization in a denaturant andremoval of the denaturant. Many proteins precipitate upon removal of thedenaturant. SDS may be used to solubilize inclusion bodies and willmaintain the proteins in solution at low concentration. However,dialysis will not always remove all of the SDS (SDS can form micelleswhich do not dialyze out); therefore, SDS-solubilized inclusion bodyprotein is soluble but not refolded.

[0091] A distinction is drawn between proteins which are soluble (i.e.,dissolved) in a solution devoid of significant amounts of ionicdetergents (e.g., SDS) or denaturants (e.g., urea, guanidinehydrochloride) and proteins which exist as a suspension of insolubleprotein molecules dispersed within the solution. A soluble protein willnot be removed from a solution containing the protein by centrifugationusing conditions sufficient to remove bacteria present in a liquidmedium (i.e., centrifugation at 12,000×g for 4-5 minutes). For example,to test whether two proteins, protein A and protein B, are soluble insolution, the two proteins are placed into a solution selected from thegroup consisting of PBS-NaCl (PBS containing 0.5 M NaCl), PBS-NaClcontaining 0.2% Tween 20, PBS, PBS containing 0.2% Tween 20, PBS-C (PBScontaining 2 mM CaCl₂), PBS-C containing either 0.1 or 0.5% Tween 20,PBS-C containing either 0.1 or 0.5% NP-40, PBS-C containing either 0.1or 0.5% Triton X-100, PBS-C containing 0.1% sodium deoxycholate. Themixture containing proteins A and B is then centrifuged at 5000×g for 5minutes. The supernatant and pellet formed by centrifugation are thenassayed for the presence of protein A and B. If protein A is found inthe supernatant and not in the pellet [except for minor amounts (i.e.,less than 10%) as a result of trapping], protein is said to be solublein the solution tested. If the majority of protein B is found in thepellet (i.e., greater than 90%), then protein B is said to exist as asuspension in the solution tested.

[0092] As used herein, the term “therapeutic arnount” refers to thatamount of antitoxin required to neutralize the pathologic effects of oneor more clostridial toxins in a subject.

[0093] The term “pyrogen” as used herein refers to a fever-producingsubstance. Pyrogens may be endogenous to the host (e.g., prostaglandins)or may be exogenous compounds (e.g., bacterial endo- and exotoxins,nonbacterial compounds such as antigens and certain steroid compounds,etc.). The presence of pyrogen in a pharmaceutical solution may bedetected using the U.S. Pharmacopeia (USP) rabbit fever test (UnitedStates Pharmacopeia, Vol. XXII (1990) United States PharmacopeialConvention, Rockville, Md., p. 151).

[0094] The term “endotoxin” as used herein refers to the high molecularweight complexes associated with the outer membrane of grain-negativebacteria. Unpurified endotoxin contains lipids, proteins andcarbohydrates. Highly purified endotoxin does not contain protein and isreferred to as lipopolysaccharide (LPS). Because unpurified endotoxin isof concern in the production of pharmaceutical compounds (e.g., proteinsproduced in E. coli using recombinant DNA technology), the termendotoxin as used herein refers to unpurified endotoxin. Bacterialendotoxin is a well known pyrogen.

[0095] As used herein, the term “endotoxin-free” when used in referenceto a composition to be administered parenterally (with the exception ofintrathecal administration) to a host means that the dose to bedelivered contains less than 5 EU/kg body weight [FDA Guidelines forParenteral Drugs (December 1987)]. Assuming a weight of 70 kg for anadult human, the dose must contain less than 350 EU to meet FDAGuidelines for parenteral administration. Endotoxin levels are measuredherein using the Limulus Amebocyte Lysate (LAL) test (Limulus AmebocyteLysate Pyrochrome™, Associates of Cape Cod, Inc. Woods Hole, Mass.). Tomeasure endotoxin levels in preparations of recombinant proteins, 0.5 mlof a solution comprising 0.5 mg of purified recombinant protein in 50 mMNaPO₄, pH 7.0, 0.3M NaCl and 10% glycerol is used in the LAL assayaccording to the manufacturer's instructions for the endpointchromogenic without diazo-coupling method [the specific components ofthe buffer containing recombinant protein to be analyzed in the LAL testare not important; any buffer having a neutral pH may be employed (seefor example, alternative buffers employed in Examples 34, 40 and 45)].Compositions containing less than or equal to than 250 endotoxin units(EU)/mg of purified recombinant protein are herein defined as“substantially endotoxin-free.” Preferably the composition contains lessthan or equal to 100, and most preferably less than or equal to 60,(EU)/mg of purified recombinant protein. Typically, administration ofbacterial toxins or toxoids to adult humans for the purpose ofvaccination involves doses of about 10-500 μg protein/dose. Therefore,administration of 10-500 μg of a purified recombinant protein to a 70 kghuman, wherein said purified recombinant protein preparation contains 60EU/mg protein, results in the introduction of only 0.6 to 30 EU (i.e.,0.2 to 8.6% of the maximum allowable endotoxin burden per parenteraldose). Administration of 10-500 μg of a purified recombinant protein toa 70 kg human, wherein said purified recombinant protein preparationcontains 250 EU/mg protein, results in the introduction of only 2.5 to125 EU (i.e., 0.7 to 36% of the maximum allowable endotoxin burden perparenteral dose).

[0096] The LAL test is accepted by the U.S. FDA as a means of detectingbacterial endotoxins (21 C.F.R. §§660.100-105). Studies have shown thatthe LAL test is equivalent or superior to the USP rabbit pyrogen testfor the detection of endotoxin and thus the LAL test can be used as asurrogate for pyrogenicity studies in animals [F. C. Perason, Pyrogens:endotoxins, LAL testing and depyrogenation, Marcel Dekker, New York(1985), pp.150-155]. The FDA Bureau of Biologics accepts the LAL assayin place of the USP rabbit pyrogen test so long as the LAL assayutilized is shown to be as sensitive as, or more sensitive as the rabbittest [Fed. Reg., 38, 26130 (1980)].

[0097] The term “monovalent” when used in reference to a clostridialvaccine refers to a vaccine which is capable of provoking an immuneresponse in a host animal directed against a single type of clostridialtoxin. For example, if immunization of a host with C. botulinum type Atoxin vaccine induces antibodies in the immunized host which protectagainst a challenge with type A toxin but not against challenge withtype B, C, D, E, F or G toxins, then the type A vaccine is said to bemonovalent. In contrast, a “multivalent” vaccine provokes an immuneresponse in a host animal directed against several (i.e., more than one)clostridial toxins. For example, if immunization of a host with avaccine comprising C. botulinum type A and B toxins induces theproduction of antibodies which protect the host against a challenge withboth type A and B toxin, the vaccine is said to be multivalent (inparticular, this hypothetical vaccine is bivalent).

[0098] As used herein the term “immunogenically-effective amount” refersto that amount of an immunogen required to invoke the production ofprotective levels of antibodies in a host upon vaccination.

[0099] The term “protective level”, when used in reference to the levelof antibodies induced upon immunization of the host with an immunogenwhich comprises a bacterial toxin, means a level of circulatingantibodies sufficient to protect the host from challenge with a lethaldose of the toxin.

[0100] As used herein the terms “protein” and “polypeptide” refer tocompounds comprising amino acids joined via peptide bonds and are usedinterchangeably.

[0101] The terms “toxin” and “neurotoxin” when used in reference totoxins produced by members (i.e., species and strains) of the genusClostridium are used interchangeably and refer to the proteins which arepoisonous to nerve tissue.

[0102] The term “receptor-binding domain” when used in reference to a C.botulinum toxin refers to the carboxy-terminal portion of the heavychain (H_(C) or the C fragment) of the toxin which is presumed to beresponsible for the binding of the active toxin (i.e., the derivativetoxin comprising the H and L chains joined via disulfide bonds) toreceptors on the surface of synaptosomes. The receptor-binding domainfor C. botulinum type A toxin is defined herein as comprising amino acidresidues 861 through 1296 of SEQ ID NO:28. The receptor-binding domainfor C. botulinum type B toxin is defined herein as comprising amino acidresidues 848 through 1291 of SEQ ID NO:40 (strain Eklund 17B). Thereceptor-binding domain of C. botulinum type C1 toxin is defined hereinas comprising amino acid residues 856 through 1291 of SEQ ID NO:60. Thereceptor-binding domain of C. botulinum type D toxin is defined hereinas comprising amino acid residues 852 through 1276 of SEQ ID NO:66. Thereceptor-binding domain of C. botulinum type E toxin is defined hereinas comprising amino acid residues 835 through 1250 of SEQ ID NO:50(Beluga strain). The receptor-binding domain of C. botulinum type Ftoxin is defined herein as comprising amino acid residues 853 through1274 of SEQ ID NO:71. The receptor-binding domain of C. botulinum type Gtoxin is defined herein as comprising amino acid residues 853 through1297 of SEQ ID NO:77. Within a given serotype, small variations in theprimary amino acid sequence of the botulinal toxins isolated fromdifferent strains has been reported [Whelan et al. (1992), supra andMinton (1995) Curr. Top. Microbiol. Immunol. 195:161-194]. The presentinvention contemplates fusion proteins comprising the receptor-bindingdomain of C. botulinum toxins from serotypes A-G including the variantsfound among different strains within a given serotype. Thereceptor-binding domains listed above are used as the prototype for eachstrain within a serotype. Fusion proteins containing an analogous regionfrom a strain other than the prototype strain are encompassed by thepresent invention.

[0103] Fusion proteins comprising the receptor binding domain (i.e., Cfragment) of botulinal toxins may include amino acid residues locatedbeyond the termini of the domains defined above. For example, thepHisBotB protein contains amino acid residues 846-1291 of SEQ ID NO:40;this fusion protein thus comprises the receptor-binding domain for C.botulinum type B toxin as defined above (i.e., Ile-848 throughGlu-1291). Similarly, pHisBotE contains amino acid residues 827-1252 ofSEQ ID NO:50 and pHisBotG contains amino acid residues 851-1297 of SEQID NO:77. Thus, both pHisBotE and pHisBotG fusion proteins contain a fewamino acids located beyond the N-terminus of the definedreceptor-binding domain.

[0104] The terms “native gene” or “native gene sequences” are used toindicate DNA sequences encoding a particular gene which contain the sameDNA sequences as found in the gene as isolated from nature. In contrast,“synthetic gene sequences” are DNA sequences which are used to replacethe naturally occurring DNA sequences when the naturally occurringsequences cause expression problems in a given host cell. For example,naturally-occurring DNA sequences encoding codons which are rarely usedin a host cell may be replaced (e.g., by site-directed mutagenesis) suchthat the synthetic DNA sequence represents a more frequently used codon.The native DNA sequence and the synthetic DNA sequence will preferablyencode the same amino acid sequence.

SUMMARY OF THE INVENTION

[0105] The present invention relates to the production of polypeptidesderived from toxins particularly in recombinant host cells. In oneembodiment, the present invention provides a host cell containing arecombinant expression vector, said vector encoding a protein comprisingat least a portion of a Clostridium botulinum toxin, said toxin selectedfrom the group consisting of type B toxin and type E toxin. The presentinvention is not limited by the nature of sequences encoding portions ofthe C. botulinum toxin. These sequences may be derived from the nativegene sequences or alternatively they may comprise synthetic genesequences. Synthetic gene sequences are employed when expression of thenative gene sequences is problematic in a given host cell (e.g., whenthe native gene sequences contain sequences resembling yeasttranscription termination signals and the desired host cell is a yeastcell).

[0106] In one embodiment, the host cell is capable of expressing therecombinant C. botulinum toxin protein at a level greater than or equalto 2% to 40% of the total cellular protein and preferably at a levelgreater than or equal to 5% of the total cellular protein. In anotherembodiment, the host cell is capable of expressing the recombinant C.botulinum toxin protein as a soluble protein at a level greater than orequal to 0.25% of the total cellular protein and preferably at a levelgreater than or equal to 0.25% to 10% of the total cellular protein.

[0107] The present invention is not limited by the nature of the hostcell employed for the production of recombinant C. botulinum toxinproteins. In a preferred embodiment, the host cell is an E. coli cell.In another preferred embodiment, the host cell is an insect cell;particularly preferred insect host cells are Spodoptera frugiperda (Sf9)cells. In another preferred embodiment, the host cell is a yeast cell;particularly preferred yeast cells are Pichia pastoris cells.

[0108] In another embodiment, the invention provides a host cellcontaining a recombinant expression vector, said vector encoding afusion protein comprising a non-toxin protein sequence and at least aportion of a Clostridium botulinum toxin, said toxin selected from thegroup consisting of type B toxin and type E toxin. The invention is notlimited by the nature of the portion of the Clostridium botulinum toxinselected. In a preferred embodiment, the portion of the toxin comprisesthe receptor binding domain (i.e., the C fragment of the toxin). Thepresent invention is not limited by the nature of the non-toxin proteinsequence employed. In a preferred embodiment, the non-toxin proteinsequence comprises a poly-histidine tract. A number of alternativefusion tags or fusion partners are known to the art (e.g., MBP, GST,protein A, etc.) and may be employed for the production of fusionproteins comprising a portion of a botulinal toxin.

[0109] The present invention further provides a vaccine comprising afusion protein, said fusion protein comprising a non-toxin proteinsequence and at least a portion of a Clostridium botulinum toxin, saidtoxin selected from the group consisting of type B toxin and type Etoxin. The vaccine may be a monovalent vaccine (i.e., containing only atoxin B fusion protein or a toxin E fusion protein), a bivalent vaccine(i.e., containing both a toxin B fusion protein and a toxin E fusionprotein) or a trivalent or higher valency vaccine. In a preferredembodiment, the toxin B fusion protein and/or toxin E fusion protein iscombined with a fusion protein comprising a non-toxin protein sequenceand at least a portion of Clostridium botulinum type A toxin. Thepresent invention is not limited by the nature of the portion of theClostridium botulinum toxin selected. In a preferred embodiment, theportion of the toxin comprises the receptor binding domain (i.e., the Cfragment of the toxin). The present invention is not limited by thenature of the non-toxin protein sequence employed. In a preferredembodiment, the non-toxin protein sequence comprises a poly-histidinetract. A number of alternative fusion tags or fusion partners are knownto the art (e.g., MBP, GST, protein A, etc.) and may be employed for thegeneration of fusion proteins comprising vaccines. When a fusion partner(i.e., the non-toxin protein sequence) is employed for the production ofa recombinant C botulinal toxin protein, the fusion partner may beremoved from the recombinant C botulinal toxin protein if desired (i.e.,prior to administration of the protein to a subject) using a variety ofmethods known to the art (e g., digestion of fusion proteins containingFactor Xa or thrombin recognition sites with the appropriate enzyme). Anumber of the pETHis vectors employed herein provide an N-terminalhis-tag followed by a Factor Xa cleavage site (see Example 28a); thebotulinal C fragment sequences follow the Factor Xa site and thus,Factor Xa can be used to remove the his-tag from the botulinal fusionprotein. In a preferred embodiment, the vaccine is substantiallyendotoxin-free.

[0110] The present invention is not limited by the method employed forthe generation of vaccine comprising fusion proteins comprising anon-toxin protein sequence and at least a portion of a Clostridiumbotulinum toxin. The fusion proteins may be produced by recombinant DNAmeans using either native or synthetic gene sequences expressed in ahost cell. The present invention is not limited to the production ofvaccines using recombinant host cells; cell free in vitrotranscription/translation systems may be employed for the expression ofthe nucleic acid constructs encoding the fusion proteins of the presentinvention. An example of such a cell-free system is the commerciallyavailable TnT™ Coupled Reticulocyte Lysate System (Promega Corporation,Madison, Wis.). Alternatively, the fusion proteins of the presentinvention may be generated by synthetic means (i.e., peptide synthesis).

[0111] The present invention further provides a method of generatingantibody directed against a Clostridium botulinum toxin comprising: a)providing in any order: i) an antigen comprising a fusion proteincomprising a non-toxin protein sequence and at least a portion of aClostridium botulinum toxin, said toxin selected from the groupconsisting of type B toxin and type E toxin, and ii) a host; and b)immunizing the host with the antigen so as to generate an antibody. In apreferred embodiment, the antigen used to immunize the host alsocontains a fusion protein comprising a non-toxin protein sequence and atleast a portion of Clostridium botulinum type A toxin. The presentinvention is not limited by the nature of the portion of the Clostridiumbotulinum toxin selected. In a preferred embodiment, the portion of thetoxin comprises the receptor binding domain (i.e., the C fragment of thetoxin). The present invention is not limited by the nature of thenon-toxin protein sequence employed. In a preferred embodiment, thenon-toxin protein sequence comprises a poly-histidine tract. A number ofalternative fusion tags or fusion partners are known to the art (e.g.,MBP, GST, protein A, etc.) and may be employed for the generation offusion proteins comprising vaccines. When a fusion partner (i.e., thenon-toxin protein sequence) is employed for the production of arecombinant C botulinal toxin protein, the fusion partner may be removedfrom the recombinant C botulinal toxin protein if desired (i.e., priorto administration of the protein to a subject) using a variety ofmethods known to the art (e.g., digestion of fusion proteins containingFactor Xa or thrombin recognition sites with the appropriate enzyme).

[0112] The present invention is not limited by the nature of the hostemployed for the production of the antibodies of the invention. In apreferred embodiment, the host is a mammal, preferably a human. Theantibodies of the present invention may be generated using non-mammalianhosts such as birds, preferably chickens. In a preferred embodiment themethod of the present invention further comprised the step c) ofcollecting the antibodies from the host. In yet another embodiment, themethod of the present invention further comprises the step d) ofpurifying the antibodies.

[0113] The present invention further provides antibodies raisedaccording to the above methods.

[0114] The present invention further contemplates multivalent vaccinescomprising at least two recombinant C. botulinum toxin proteins derivedfrom the group consisting of C. botulinum serotypes A, B, C, D, E, F,and G. The invention contemplates bivalent, trivalent, quadravalent,pentavalent, heptavalent and septivalent vaccines comprising recombinantC. botulinum toxin proteins. Preferably the recombinant C. botulinumtoxin protein comprises the receptor binding domain (i.e., C fragment)of the toxin.

DESCRIPTION OF THE INVENTION

[0115] The present invention contemplates vaccinating humans and otheranimals with polypeptides derived from C. botulinum neurotoxins whichare substantially endotoxin-free. These botulinal peptides are alsouseful for the production of antitoxin. Anti-botulinal toxin antitoxinis useful for the treatment of patients effected by or at risk ofsymptoms due to the action of C. botulinum toxins. The organisms, toxinsand individual steps of the present invention are described separatelybelow.

[0116] I. Clostridium Species, Clostridial Diseases And AssociatedToxins

[0117] A preferred embodiment of the method of the present invention isdirected toward obtaining antibodies against Clostridium species, theirtoxins, enzymes or other metabolic by-products, cell wall components, orsynthetic or recombinant versions of any of these compounds. It iscontemplated that these antibodies will be produced by immunization ofhumans or other animals. It is not intended that the present inventionbe limited to any particular toxin or any species of organism. In oneembodiment, toxins from all Clostridium species are contemplated asimmunogens. Examples of these toxins include the neuramimidase toxin ofC. butyricum, C. sordellii toxins HT and LT, toxins A, B, C, D, E, F,and G of C. botulinum and the numerous C. perfringens toxins. In onepreferred embodiment, toxins A, B, and E of C. botulinum arecontemplated as immunogens. Table 2 above lists various Clostridiumspecies, their toxins and some antigens associated with disease. TABLE 2Clostridial Toxins Organism Toxins and Disease-Associated Antigens C.botulinum A, B, C₁, C₂, D, E, F, G C. butyricum Neuraminidase C.difficile A, B, Enterotoxin (not A nor B), Motility Altering Factor, LowMolecular Weight Toxin, Others C. perfringens α, β, ε, ι, γ, δ, ν, θ, κ,λ, μ, υ C. sordelli/ HT, LT, α, β, γ C. bifermentans C. novyi α, β, γ,δ, ε, ζ, ν, θ C. septicum α, β, γ, δ C. histolyticum α, β, γ, δ, ε plusadditional enzymes C. chauvoei α, β, γ, δ

[0118] It is not intended that antibodies produced against one toxinwill only be used against that toxin. It is contemplated that antibodiesdirected against one toxin (e.g., C. perfringens type A enterotoxin) maybe used as an effective therapeutic against one or more toxin(s)produced by other members of the genus Clostridium or other toxinproducing organisms (e.g., Bacillus cereus, Staphylococcus aureus,Streptococcus mutans, Acinetobacter calcoaceticus, Pseudomonasaeruginosa, other Pseudomonas species, etc.). It is further contemplatedthat antibodies directed against the portion of the toxin which binds tomammalian membranes (e.g., C. perfringens enterotoxin A) can also beused against other organisms. It is contemplated that these membranebinding domains are produced synthetically and used as immunogens.

[0119] II. Obtaining Antibodies in Non-Mammals

[0120] A preferred embodiment of the method of the present invention forobtaining antibodies involves immunization. However, it is alsocontemplated that antibodies could be obtained from non-mammals withoutimmunization. In the case where no immunization is contemplated, thepresent invention may use non-mammals with preexisting antibodies totoxins as well as non-mammals that have antibodies to whole organisms byvirtue of reactions with the administered antigen. An example of thelatter involves immunization with synthetic peptides or recombinantproteins sharing epitopes with whole organism components.

[0121] In a preferred embodiment, the method of the present inventioncontemplates immunizing non-mammals with bacterial toxin(s). It is notintended that the present invention be limited to any particular toxin.In one embodiment, toxin from all clostridial bacteria sources (seeTable 2) are contemplated as immunogens. Examples of these toxins are C.butyricum neuramimidase toxin, toxins A, B, C, D, E, F, and G from C.botulinum, C. perfringens toxins α, β, ε, and ι, and C. sordellii toxinsHT and LT. In a preferred embodiment, C. botulinum toxins A, B, C, D, E,and F (or fragments thereof) are contemplated as immunogens.

[0122] A particularly preferred embodiment involves the use of bacterialtoxin protein or fragments of toxin proteins produced by molecularbiological means (i.e., recombinant toxin proteins). In a preferredembodiment, the immunogen comprises the receptor-binding domain (i.e.,the ˜50 kD carboxy-terminal portion of the heavy chain; also referred toas the C fragment) of C. botulinum serotype A neurotoxin produced byrecombinant DNA technology. In another preferred embodiment, theimmunogen comprises the receptor-binding domain of C. botulinum serotypeB neurotoxin produced by recombinant DNA technology. In yet anotherpreferred embodiment, the immunogen comprises the receptor-bindingdomain region of C. botulinum serotype E neurotoxin produced byrecombinant DNA technology. In yet another preferred embodiment, theimmunogen comprises the receptor-binding domain region of C. botulinumserotype C1 neurotoxin produced by recombinant DNA technology. In yetanother preferred embodiment, the immunogen comprises thereceptor-binding domain region of C. botulinum serotype C2 neurotoxinproduced by recombinant DNA technology. In yet another preferredembodiment, the immunogen comprises the receptor-binding domain regionof C. botulinum serotype D neurotoxin produced by recombinant DNAtechnology. In yet another preferred embodiment, the immunogen comprisesthe receptor-binding domain region of C. botulinum serotype F neurotoxinproduced by recombinant DNA technology. In yet another preferredembodiment, the immunogen comprises the receptor-binding domain regionof C. botulinum serotype G neurotoxin produced by recombinant DNAtechnology. In a preferred embodiment, the recombinant botulinal toxinproteins are expressed as fusion proteins (e.g., as histidine-taggedproteins). In a still further preferred embodiment, the immunogen is amultivalent vaccine comprising the receptor-binding domain region of C.botulinum toxin from two or more toxins selected from the groupconsisting of type A, type B, type C (including C1 and C2), type D, typeE, and type F toxin.

[0123] When immunization is used, the preferred non-mammal is from theclass Aves. All birds are contemplated (e.g., duck, ostrich, emu,turkey, etc.). A preferred bird is a chicken. Importantly, chickenantibody does not fix mammalian complement. [See H. N. Benson et al., J.Immunol. 87:616 (1961).] Thus, chicken antibody will normally not causea complement-dependent reaction. [A. A. Benedict and K. Yamaga,“Immunoglobulins and Antibody Production in Avian Species,” inComparative Immunology (J. J. Marchaloni, ed.), pp. 335-375, Blackwell,Oxford (1966).] Thus, the preferred antitoxins of the present inventionwill not exhibit complement-related side effects observed withantitoxins known presently.

[0124] When birds are used, it is contemplated that the antibody will beobtained from either the bird serum or the egg. A preferred embodimentinvolves collection of the antibody from the egg. Laying hens transportimmunoglobulin to the egg yolk (“IgY”) in concentrations equal to orexceeding that found in serum. [See R. Patterson et al., J. Immunol.89:272 (1962); and S. B. Carroll and B. D. Stollar, J. Biol. Chem.258:24 (1983).] In addition, the large volume of egg yolk producedvastly exceeds the volume of serum that can be safely obtained from thebird over any given time period. Finally, the antibody from eggs ispurer and more homogeneous; there is far less non-immunoglobulin protein(as compared to serum) and only one class of immunoglobulin istransported to the yolk.

[0125] When considering immunization with toxins, one may considermodification of the toxins to reduce the toxicity. In this regard, it isnot intended that the present invention be limited by immunization withmodified toxin. Unmodified (“native”) toxin is also contemplated as animmunogen.

[0126] It is also not intended that the present invention be limited bythe type of modification—if modification is used. The present inventioncontemplates all types of toxin modification, including chemical andheat treatment of the toxin. The preferred modification, however, isformaldehyde treatment.

[0127] It is not intended that the present invention be limited to aparticular mode of immunization; the present invention contemplates allmodes of immunization, including subcutaneous, intramuscular,intraperitoneal, and intravenous or intravascular injection, as well asper os administration of immunogen.

[0128] The present invention further contemplates immunization with orwithout adjuvant. (Adjuvant is defined as a substance known to increasethe immune response to other antigens when administered with otherantigens.) If adjuvant is used, it is not intended that the presentinvention be limited to any particular type of adjuvant—or that the sameadjuvant, once used, be used all the time. While the present inventioncontemplates all types of adjuvant, whether used separately or incombinations, the preferred use of adjuvant is the use of CompleteFreund's Adjuvant followed sometime later with Incomplete Freund'sAdjuvant. Another preferred use of adjuvant is the use of GerbuAdjuvant. The invention also contemplates the use of RIBI fowl adjuvantand Quil A adjuvant.

[0129] When immunization is used, the present invention contemplates awide variety of immunization schedules. In one embodiment, a chicken isadministered toxin(s) on day zero and subsequently receives toxin(s) inintervals thereafter. It is not intended that the present invention belimited by the particular intervals or doses. Similarly, it is notintended that the present invention be limited to any particularschedule for collecting antibody. The preferred collection time issometime after day 100.

[0130] Where birds are used and collection of antibody is performed bycollecting eggs, the eggs may be stored prior to processing forantibody. It is preferred that eggs be stored at 4° C. for less than oneyear.

[0131] It is contemplated that chicken antibody produced in this mannercan be buffer-extracted and used analytically. While unpurified, thispreparation can serve as a reference for activity of the antibody priorto further manipulations (e.g., immunoaffinity purification).

[0132] III. Increasing the Effectiveness of Antibodies

[0133] When purification is used, the present invention contemplatespurifying to increase the effectiveness of both non-mammalian antitoxinsand mammalian antitoxins. Specifically, the present inventioncontemplates increasing the percent of toxin-reactive immunoglobulin.The preferred purification approach for avian antibody is polyethyleneglycol (PEG) separation.

[0134] The present invention contemplates that avian antibody beinitially purified using simple, inexpensive procedures. In oneembodiment, chicken antibody from eggs is purified by extraction andprecipitation with PEG. PEG purification exploits the differentialsolubility of lipids (which are abundant in egg yolks) and yolk proteinsin high concentrations of PEG 8000. [Polson et al., Immunol. Comm. 9:495(1980).] The technique is rapid, simple, and relatively inexpensive andyields an immunoglobulin fraction that is significantly purer in termsof contaminating non-immunoglobulin proteins than the comparableammonium sulfate fractions of mammalian sera and horse antibodies. Themajority of the PEG is removed from the precipitated chickenimmunoglobulin by treatment with ethanol. Indeed, PEG-purified antibodyis sufficiently pure that the present invention contemplates the use ofPEG-purified antitoxins in the passive immunization of intoxicatedhumans and animals.

[0135] IV. Treatment The present invention contemplates antitoxintherapy for humans and other animals intoxicated by bacterial toxins. Apreferred method of treatment is by intravenous administration ofanti-boutlinal antitoxin; oral administration is also contemplated forother clostridial antitoxins.

[0136] A. Dosage of Antitoxin

[0137] It was noted by way of background that a balance must be struckwhen administering currently available antitoxin which is usuallyproduced in large animals such as horses; sufficient antitoxin must beadministered to neutralize the toxin, but not so much antitoxin as toincrease the risk of untoward side effects. These side effects arecaused by: i) patient sensitivity to foreign (e.g, horse) proteins; ii)anaphylactic or immunogenic properties of non-immunoglobulin proteins;iii) the complement fixing properties of mammalian antibodies; and/oriv) the overall burden of foreign protein administered. It is extremelydifficult to strike this balance when, as noted above, the degree ofintoxication (and hence the level of antitoxin therapy needed) can onlybe approximated.

[0138] The present invention contemplates significantly reducing sideeffects so that this balance is more easily achieved. Treatmentaccording to the present invention contemplates reducing side effects byusing PEG-purified antitoxin from birds.

[0139] In one embodiment, the treatment of the present inventioncontemplates the use of PEG-purified antitoxin from birds. The use ofyolk-derived, PEG-purified antibody as antitoxin allows for theadministration of: 1) non(mammalian)-complement-fixing, avian antibody;2) a less heterogeneous mixture of non-immunoglobulin proteins; and 3)less total protein to deliver the equivalent weight of active antibodypresent in currently available antitoxins. The non-mammalian source ofthe antitoxin makes it useful for treating patients who are sensitive tohorse or other mammalian sera.

[0140] B. Delivery of Antitoxin

[0141] Although it is not intended to limit the route of delivery, thepresent invention contemplates a method for antitoxin treatment ofbacterial intoxication in which delivery of antitoxin is oral. In oneembodiment, antitoxin is delivered in a solid form (e.g., tablets). Inan alternative embodiment antitoxin is delivered in an aqueous solution.When an aqueous solution is used, the solution has sufficient ionicstrength to solubilize antibody protein, yet is made palatable for oraladministration. The delivery solution may also be buffered (e.g.,carbonate buffer pH 9.5) which can neutralize stomach acids andstabilize the antibodies when the antibodies are administered orally. Inone embodiment the delivery solution is an aqueous solution. In anotherembodiment the delivery solution is a nutritional formula. Preferably,the delivery solution is infant formula. Yet another embodimentcontemplates the delivery of lyophilized antibody encapsulated ormicroencapsulated inside acid-resistant compounds.

[0142] Methods of applying enteric coatings to pharmaceutical compoundsare well known to the art [companies specializing in the coating ofpharmaceutical compounds are available; for example, The Coating Place(Verona, Wis.) and AAI (Wilmington, N.C.)]. Enteric coatings which areresistant to gastric fluid and whose release (i.e., dissolution of thecoating to release the pharmaceutical compound) is pH dependent arecommercially available [for example, the polymethacrylates Eudragit® Land Eudragit® S (Röhm GmbH)]. Eudragit® S is soluble in intestinal fluidfrom pH 7.0; this coating can be used to microencapsulate lyophilizedantitoxin antibodies and the particles are suspended in a solutionhaving a pH above or below pH 7.0 for oral administration. Themicroparticles will remain intact and undissolved until they reached theintestines where the intestinal pH would cause them to dissolve therebyreleasing the antitoxin.

[0143] The invention contemplates a method of treatment which can beadministered for treatment of acute intoxication. In one embodiment,antitoxin is administered orally in either a delivery solution or intablet form, in therapeutic dosage, to a subject intoxicated by thebacterial toxin which served as immunogen for the antitoxin.

[0144] The invention also contemplates a method of treatment which canbe administered prophylactically. In one embodiment, antitoxin isadministered orally, in a delivery solution, in therapeutic dosage, to asubject, to prevent intoxication of the subject by the bacterial toxinwhich served as immunogen for the production of antitoxin. In anotherembodiment, antitoxin is administered orally in solid form such astablets or as microencapsulated particles. Microencapsulation oflyophilized antibody using compounds such as Eudragit® (Rohm GmbH) orpolyethylene glycol, which dissolve at a wide range of pH units, allowsthe oral administration of solid antitoxin in a liquid form (i.e., asuspension) to recipients unable to tolerate administration of tablets(e.g., children or patients on feeding tubes). In one preferredembodiment the subject is a child. In another embodiment, antibodyraised against whole bacterial organism is administered orally to asubject, in a delivery solution, in therapeutic dosage.

[0145] V. Vaccines Against Clostridial Species

[0146] The invention contemplates the generation of mono- andmultivalent vaccines for the protection of an animal (particularlyhumans) against several clostridial species. Of particular interest arevaccines which stimulate the production of a humoral immune response toC. botulinum, C. tetani and C. difficile in humans. The antigenscomprising the vaccine preparation may be native or recombinantlyproduced toxin proteins from the clostridial species listed above. Whentoxin proteins are used as immunogens they are generally modified toreduce the toxicity. This modification may be by chemical or genetic (ie., recombinant DNA technology) means. In general genetic detoxification(i.e., the expression of nontoxic fragments in a host cell) is preferredas the expression of nontoxic fragments in a host cell precludes thepresence of intact, active toxin in the final preparation. However, whenchemical modification is desired, the preferred toxin modification isformaldehyde treatment.

[0147] The invention contemplates that recombinant C. botulinum toxinproteins be used as antigens in mono- and multivalent vaccinepreparations. Soluble, substantially endotoxin-free recombinant C.botulinum toxin proteins derived from serotypes A, B and E may be usedindividually (i.e., as mono-valent vaccines) or in combination (i.e., asa multi-valent vaccine). In addition, the recombinant C. botulinum toxinproteins derived from serotpes A, B and E may be used in conjunctionwith either recombinant or native toxins or toxoids from other serotypesof C. botulinum, C. difficile and C. tetani as antigens for thepreparation of these mono- and multivalent vaccines. It is contemplatedthat, due to the structural similarity of C. botulinum and C. tetanitoxin proteins, a vaccine comprising C. difficile and botulinum toxinproteins (native or recombinant or a mixture thereof) be used tostimulate an immune response against C. botulinum, C. tetani and C.difficile.

[0148] The present invention further contemplates multi-valent vaccinescomprising two or more botulinal toxin proteins selected from the groupcomprising recombinant C. botulinum toxin proteins derived fromserotypes A, B, C (including C1 and C2), D, E, F and G.

[0149] The adverse consequences of exposure to botulinal toxin would beavoided by immunization of subjects at risk of exposure to the toxinwith nontoxic preparations which confer immunity such as chemically orgenetically detoxified toxin.

[0150] Vaccines which confer immunity against one or more of the toxintypes A, B, E, F and G would be useful as a means of protecting humansfrom the deleterious effects of those C. botulinum toxins known toaffect man. Indeed as the possibility exists that humans could beexposed to any of the seven serotypes of C. botulinum toxin (e.g.,during biological warfare or the production of toxin in a laboratorysetting), multivalent vaccines capable of conferring immunity againsttoxin types A-G (including both C1 and C2 toxins) would be useful forthe protection of humans. Vaccines which confer immunity against one ormore of the toxin types C, D and E would be useful for veterinaryapplications.

[0151] The botulinal neurotoxin is synthesized as a single polypeptidechain which is processed into a heavy (H; ˜100 kD) and a light (L; ˜50kD) chain by cleavage with proteolytic enzymes; these two chains areheld together via disulfide bonds in the active toxin (referred to asderivative toxin) [B. R. DasGupta and H. Sugiyama, Biochem. Biophys.Res. Commun. 48:108 (1972); reviewed in B. R. DasGupta, J. Physiol.84:220 (1990), H. Sugiyama, Microbiol. Rev. 44:419 (1980) and C. L.Hatheway, Clin. Microbiol. Rev. 3:66 (1990)]. The heavy chain of theactive toxin is cleaved by trypsin to produce two fragments termed H_(C)(also referred to as H₁ or C) and H_(N) (also referred to as H₂ or B).The H_(C) fragment (46 kD) comprises the carboxy end of the H chain. TheH_(N) fragment (˜49 kD) comprises the animo end and remains attached tothe L chain (H_(N)L). Neither H_(C) or H_(N)L is toxic. H_(C) competeswith whole derivative toxin for binding to synaptosomes and thereforeH_(C) is said to contain the receptor binding site. The H_(C) and H_(N)fragments of botulinal toxin are analogous to the fragments C and B oftetanus toxin which are produced by papain cleavage. The C fragment oftetanus toxin has been shown to be responsible for the binding oftetanus toxin to purified gangliosides and neuronal cells [Halpern andLoftus, J. Biol. Chem. 288:11188 (1993)].

[0152] Antisera raised against purified preparations of isolatedbotulinal H and L chains have been shown to protect mice against thelethal effects of the toxin; however, the effectiveness of the twoantisera differ with the anti-H sera being more potent (H. Sugiyama,supra). While the different botulinal toxins show structural similarityto one another, the different serotypes are reported to beimmunologically distinct (i.e., sera raised against one toxin type doesnot cross-react to a significant degree with other types). Thus, thegeneration of multivalent vaccines may require the use of more than onetype of toxin.

[0153]C. botulinum toxin genes from all seven serotypes have been clonedand sequenced (Minton (1995), supra); in addition, partial amino acidsequence is available for a number of C. botulinum toxins isolated fromdifferent strains within a given serotype. The C. botulinum toxinscontain about 1250-1300 amino acid residues. On the DNA level, theoverall degree of homology between C. botulinum serotypes A, B, C, D andE toxins averages between 50 and 60% identity with a greater degree ofhomology being found between H chain-encoding regions than between thoseencoding L chains [Whelan et al. (1992) Appl. Environ. Microbiol.58:2345]. The degree of identity between C. botulinum toxins on theamino acid level reflects the level of DNA sequence homology. The mostdivergent area of DNA and amino acid sequence is found within thecarboxy-terminal area of the various C. botulinum H chain genes. Thisportion of the toxin (i.e., H_(C) or the C fragment) plays a major rolein cell binding. As toxin from different serotypes is thought to bind todistinct cell receptor molecules, it is not surprising that the toxinsdiverge significantly over this region.

[0154] Within a given serotype, small variations in the primary aminoacid sequence of the botulinal toxins isolated from different strainshas been reported [Whelan et al. (1992), supra and Minton (1995),supra]. The present invention contemplates fusion proteins comprisingportions of C. botulinum toxins from serotypes A-G including thevariants found among different strains within a given serotype. Thepresent invention provides oligonucleotide primers which may be used toamplify the C fragment or receptor-binding region of the toxin gene fromvarious strains of C. botulinum serotype A, serotype B, serotype C(C1and C2), serotype D, serotype E, serotype F and serotype G. A largenumber of different strains of C. botulinum serotype A, serotype B,serotype C, serotype D serotype E and serotype F are available from theAmerican Type Culture Collection (ATCC; Rockville, Md.). For example,the ATCC provides the following: Type A strains: 174 (ATCC 3502), 457(ATCC 17862), and NCTC 7272 (ATCC 19397); Type B strains: 34 (ATCC 439),62A (ATCC 7948), NCA 213 B (ATCC 7949), 13114 (ATCC 8083), 3137 (ATCC17780), 1347 (ATCC 17841), 2017 (ATCC 17843), 2217 (ATCC 17844), 2254(ATCC 17845) and VP 1731 (ATCC 25765); Type C strains: 2220 (ATCC17782), 2239 (ATCC 17783), 2223 (ATCC 17784; a type C-β strain; C-βstrains produce C2 toxin), 662 (ATCC 17849; a type C-α strain; C-αstrains produce mainly C1 toxin and a small amount of C2 toxin), 2021(ATCC 17850; a type C-α strain) and VPI 3803 (ATCC 25766); Type Dstrains: ATCC 9633, 2023 (ATCC 17851), and VPI 5995 (ATCC 27517); Type Estrains: ATCC 43181, 36208 (ATCC 9564), 2231 (ATCC 17786), 2229 (ATCC17852), 2279 (ATCC 17854) and 2285 (ATCC 17855) and Type F strains: 202F(ATCC 23387), VPI 4404 (ATCC 25764), VPI 2382 (ATCC 27321) and Langeland(ATCC 35415). Type G strain, 113/30 (NCFB 3012) may be obtained from theNational Collection of Food Bacteria (NCFB, AFRC Institute of FoodResearch, Reading, United Kingdom).

[0155] Purification methods have been reported for native toxin types A,B, C, D, E, and F [reviewed in G. Sakaguchi, Pharmac. Ther. 19:165(1983)]. As the different botulinal toxins are structurally related, theinvention contemplates the expression of any of the botulinal toxins(e.g, types A-G) as soluble recombinant fusion proteins.

[0156] In particular, methods for purification of the type A botulinumneurotoxin have been developed [L. J. Moberg and H. Sugiyama, Appl.Environ. Microbiol. 35:878 (1978)]. Immunization of hens with detoxifiedpurified protein results in the generation of neutralizing antibodies[B. S. Thalley et al., in Botulinum and Tetanus Neurotoxins, B. R.DasGupta, ed., Plenum Press, New York (1993), p. 467].

[0157] The currently available C. botulinum pentavalent vaccinecomprising chemically inactivated (i.e., formaldehyde treated) type A,B, C, D and E toxins is not adequate. The efficacy is variable (inparticular, only 78% of recipients produce protective levels ofanti-type B antibodies following administration of the primary series)and immunization is painful (deep subcutaneous inoculation is requiredfor administration), with adverse reactions being common (moderate tosevere local reactions occur in approximately 6% of recipients uponinitial injection; this number rises to approximately 11% of individualswho receive booster injections) [Informational Brochure for thePentavalent (ABCDE) Botulinum Toxoid, Centers for Disease Control].Preparation of this vaccine is dangerous as active toxin must be handledby laboratory workers.

[0158] In general, chemical detoxification of bacterial toxins usingagents such as formaldehyde, glutaraldehyde or hydrogen peroxide is notoptimal for the generation of vaccines or antitoxins. A delicate balancemust be struck between too much and too little chemical modification. Ifthe treatment is insufficient, the vaccine may retain residual toxicity.If the treatment is too excessive, the vaccine may lose potency due todestruction of native immunogenic determinants. Another major limitationof using botulinal toxoids for the generation of antitoxins or vaccinesis the high production expense. For the above reasons, the developmentof methods for the production of nontoxic but immunogenic C. botulinumtoxin proteins is desirable.

[0159] The C. botulinum and C tetanus toxin proteins have similarstructures [reviewed in E. J. Schantz and E. A. Johnson, Microbiol. Rev.56:80 (1992)]. The carboxy-terminal 50 kD fragment of the tetanus toxinheavy chain (fragment C) is released by papain cleavage and has beenshown to be non-toxic and immunogenic. Recombinant tetanus toxinfragment C has been developed as a candidate vaccine antigen [A. J.Makoff et al., Bio/Technology 7:1043 (1989)]. Mice immunized withrecombinant tetanus toxin fragment C were protected from challenge withlethal doses of tetanus toxin. No studies have demonstrated that therecombinant tetanus fragment C protein confers immunity against otherbotulinal toxins such as the C. botulinum toxins.

[0160] Recombinant tetanus fragment C has been expressed in E. coli (A.J. Makoff et al., Bio/Technology, supra and Nucleic Acids Res. 17:10191(1989); J. L. Halpern et al., Infect. Immun. 58:1004 (1990)], yeast [M.A. Romanos et al., Nucleic Acids Res. 19:1461 (1991)] and baculovirus[I. G. Charles et al., Infect. Immun. 59:1627 (1991)]. Synthetic tetanustoxin genes had to be constructed to facilitate expression in yeast (M.A. Romanos et al., supra) and E. coli [A. J. Makoff et al., NucleicAcids Res., supra], due to the high A-T content of the tetanus toxingene sequences. High A-T content is a common feature of clostridialgenes (M. R. Popoff et al., Infect. Immun. 59:3673 (1991); H. F.LaPenotiereu et al., in Botulinum and Tetanus Neurotoxins, B. R.DasGupta, ed., Plenum Press, New York (1993), p. 463] which createsexpression difficulties in E. coli and yeast due primarily to alteredcodon usage frequency and fortuitous polyadenylation sites,respectively.

[0161] The C fragment of the C. botulinum type A neurotoxin heavy chainhas been evaluated as a vaccine candidate. The C. botulinum type Aneurotoxin gene has been cloned and sequenced [D. E. Thompson et al.,Eur. J. Biochem. 189:73 (1990)]. The C fragment of the type A toxin wasexpressed as either a fusion protein comprising the botulinal C fragmentfused with the maltose binding protein (MBP) or as a native protein [H.F. LaPenotiere et al, (1993) supra, H. F. LaPenotiere et al., Toxicon.33:1383 (1995) and Middlebrook and Brown (1995), Curr. Top. Microbiol.Immunol. 195:89-122]. The plasmid construct encoding the native proteinwas reported to be unstable, while the fusion protein was expressedprimarily in inclusion bodies as insoluble protein. Immunization of micewith crudely purified MBP fusion protein resulted in protection againstIP challenge with 3 LD₅₀ doses of toxin [LaPenotiere et al., (1993) and(1995), supra]. However, this recombinant C. botulinum type A toxin Cfragment/MBP fusion protein is not a suitable immunogen for theproduction of vaccines as it is expressed as an insoluble protein in E.coli. Furthermore, this recombinant C. botulinum type A toxin Cfragment/BP fusion protein was not shown to be substantially free ofendotoxin contamination. Experience with recombinant C. botulinum type Atoxin C fragment/MBP fusion proteins shows that the presence of the MBPon the fusion protein greatly complicates the removal of endotoxin frompreparations of the recombinant fusion protein (see Ex. 24, infra).Expression of a synthetic gene encoding C. botulinum type A toxin Cfragment as a soluble protein excreted from insect cells has beenreported [Middlebrook and Brown (1995), supra]; no details regarding thelevel of expression achieved or the presence of endotoxin or otherpyrogens were provided. Like the insoluble protein expressed in E. coli,immunization with the recombinant protein produced in insect cells wasreported to protect mice from challenge with C. botulinum toxin A.

[0162] Inclusion body protein must be solubilized prior to purificationand/or administration to a host. The harsh treatment of inclusion bodyprotein needed to accomplish this solubilization may reduce theimmunogenicity of the purified protein. Ideally, recombinant proteins tobe used as vaccines are expressed as soluble proteins at high levels(i.e., greater than or equal to about 0.75% of total cellular protein)in E. coli or other host cells (e.g., yeast, insect cells, etc.). Thisfacilitates the production and isolation of sufficient quantities of theimmunogen in a highly purified form (i.e., substantially free ofendotoxin or other pyrogen contamination). The ability to expressrecombinant toxin proteins as soluble proteins in E. coli isadvantageous due to the low cost of growth compared to insect ormammalian tissue culture cells.

[0163] The C. botulinum type B neurotoxin gene has been cloned andsequenced from two strains of C. botulinum type B [Whelan et al. (1992)Appl. Environ. Microbiol. 58:2345 (Danish strain) and Hutson et al.(1994) Curr. Microbiol. 28:101 (Eklund 17B strain)]. The nucleotidesequence of the toxin gene derived from the Eklund 17B strain (ATCC25765) is available from the EMBL/GemBank sequence data banks under theaccession number X71343; the nucleotide sequence of the coding region islisted in SEQ ID NO:39. The amino acid sequence of the C. botulinum typeB neurotoxin derived from the strain Eklund 17B is listed in SEQ IDNO:40. The nucleotide sequence of the C. botulinum serotype B toxin genederived from the Danish strain is listed in SEQ ID NO:41. The amino acidsequence of the C. botulinum type B neurotoxin derived from the Danishstrain is listed in SEQ ID NO:42.

[0164] The C. botulinum type B neurotoxin gene is synthesized as asingle polypeptide chain which is processed to form a dimer composed ofa light and a heavy chain linked via disulfide bonds. The light chain isresponsible for pharmacological activity (i.e., inhibition of therelease of acetylcholine at the neuromuscular junction). The N-terminalportion of the heavy chain is thought to mediate channel formation whilethe C-terminal portion mediates toxin binding; the type B neurotoxin hasbeen reported to exist as a mixture of predominantly single chain withsome double chain (Whelan et al., supra). The 50 kD carboxy-terminalportion of the heavy chain is referred to as the C fragment or the H_(C)domain. The present invention reports for the first time, the expressionof the C fragment of C. botulinum type B toxin in heterologous hosts(e.g., E. coli).

[0165] The C. botulinum type E neurotoxin gene has been cloned andsequenced from a number of different strains [Poulet et al. (1992)Biochem. Biophys. Res. Commun. 183:107; Whelan et al. (1992) Eur. J.Biochem. 204:657; and Fujii et al. (1993) J. Gen. Microbiol. 139:79].The nucleotide sequence of the type E toxin gene is available from theEMBL sequence data bank under accession numbers X62089 (strain Beluga)and X62683 (strain NCTC 11219); the nucleotide sequence of the codingregion (strain Beluga) is listed in SEQ ID NO:45. The amino acidsequence of the C. botulinum type E neurotoxin derived from strainBeluga is listed in SEQ ID NO:46. The type E neurotoxin gene issynthesized as a single polypeptide chain which may be converted to adouble-chain form (i.e., a heavy chain and a light chain) by cleavagewith trypsin; unlike the type A neurotoxin, the type E neurotoxin existsessentially only in the single-chain form. The 50 kD carboxy-terminalportion of the heavy chain is referred to as the C fragment or the H_(C)domain. The present invention reports for the first time, the expressionof the C fragment of C. botulinum type E toxin in heterologous hosts(e.g., E. coli).

[0166] The C. botulinum type C1, D, F and G neurotoxin genes have beencloned and sequenced. The nucleotide and amino acid sequences of thesegenes and toxins are provided herein. The invention provides methods forthe expression of the C fragment from each of these toxin genes inheterologous hosts and the purification of the resulting recombinantproteins.

[0167] The subject invention provides methods which allow the productionof soluble C. botulinum toxin proteins in economical host cells (e.g., Ecoli). In addition the subject invention provides methods which allowthe production of soluble botulinal toxin proteins in yeast and insectcells. Further, methods for the isolation of purified soluble C.botulinum toxin proteins which are suitable for immunization of humansand other animals are provided. These soluble, purified preparations ofC. botulinum toxin proteins provide the basis for improved vaccinepreparations and facilitate the production of antitoxin.

[0168] When recombinant clostridial toxin proteins produced ingram-negative bacteria (e.g., E. coli) are used as vaccines, they arepurified to remove endotoxin prior to administration to a host animal.In order to vaccinate a host, an immunogenically-effective amount ofpurified substantially endotoxin-free recombinant clostridial toxinprotein is administered in any of a number of physiologically acceptablecarriers known to the art. When administered for the purpose ofvaccination, the purified substantially endotoxin-free recombinantclostridial toxin protein may be used alone or in conjunction with knownadjutants, including potassium alum, aluminum phosphate, aluminumhydroxide, Gerbu adjuvant (GMDP; C.C. Biotech Corp.), RIBI adjuvant(MPL; RIBI Immunochemical Research, Inc.), QS21 (Cambridge Biotech). Thealum and aluminum-based adjutants are particularly preferred whenvaccines are to be administered to humans; however, any adjuvantapproved for use in humans may be employed. The route of immunizationmay be nasal, oral, intramuscular, intraperitoneal or subcutaneous.

[0169] The invention contemplates the use of soluble, substantiallyendotoxin-free preparations of fusion proteins comprising the C fragmentof the C. botulinum type A, B, C, D, E, F, and G toxin as vaccines. Inone embodiment, the vaccine comprises the C fragment of either the C.botulinum type A, B, C, D, E, F, or G toxin and a poly-histidine tract(also called a histidine tag). In a particularly preferred embodiment, afusion protein comprising the histidine tagged C fragment is expressedusing the pET series of expression vectors (Novagen). The pET expressionsystem utilizes a vector containing the T7 promoter which encodes thefusion protein and a host cell which can be induced to express the T7DNA polymerase (i.e., a DE3 host strain). The production of C fragmentfusion proteins containing a histidine tract is not limited to the useof a particular expression vector and host strain. Several commerciallyavailable expression vectors and host strains can be used to express theC fragment protein sequences as a fusion protein containing a histidinetract (For example, the pQE series (pQE-8, 12, 16, 17, 18, 30, 31, 32,40, 41, 42, 50, 51, 52, 60 and 70) of expression vectors (Qiagen) whichare used with the host strains M15[pREP4] (Qiagen) and SG13009[pREP4](Qiagen) can be used to express fusion proteins containing six histidineresidues at the amino-terminus of the fusion protein). Furthermore anumber of commercially available expression vectors which provide ahistidine tract also provide a protease cleavage site between thehistidine tract and the protein of interest (e.g., botulinal toxinsequences). Cleavage of the resulting fusion protein with theappropriate protease will remove the histidine tag from the protein ofinterest (e.g., botulinal toxin sequences) (see Example 28a, infra).Removal of the histidine tag may be desirable prior to administration ofthe recombinant botulinal toxin protein to a subject (e.g., a human).

[0170] VI. Detection of Toxin

[0171] The invention contemplates detecting bacterial toxin in a sample.The term “sample” in the present specification and claims is used in itsbroadest sense. On the one hand it is meant to include a specimen orculture. On the other hand, it is meant to include both biological andenvironmental samples.

[0172] Biological samples may be animal, including human, fluid, solid(e.g., stool) or tissue; liquid and solid food products and ingredientssuch as dairy items, vegetables, meat and meat by-products, and waste.Environmental samples include environmental material such as surfacematter, soil, water and industrial samples, as well as samples obtainedfrom food and dairy processing instruments, apparatus, equipment,disposable and non-disposable items. These examples are not to beconstrued as limiting the sample types applicable to the presentinvention.

[0173] The invention contemplates detecting bacterial toxin by acompetitive immunoassay method that utilizes recombinant toxin A andtoxin B proteins, antibodies raised against recombinant bacterial toxinproteins. A fixed amount of the recombinant toxin proteins areimmobilized to a solid support (e.g., a microtiter plate) followed bythe addition of a biological sample suspected of containing a bacterialtoxin. The biological sample is first mixed with affinity-purified orPEG fractionated antibodies directed against the recombinant toxinprotein. A reporter reagent is then added which is capable of detectingthe presence of antibody bound to the immobilized toxin protein. Thereporter substance may comprise an antibody with binding specificity forthe antitoxin attached to a molecule which is used to identify thepresence of the reporter substance. If toxin is present in the sample,this toxin will compete with the immobilized recombinant toxin proteinfor binding to the anti-recombinant antibody thereby reducing the signalobtained following the addition of the reporter reagent. A control isemployed where the antibody is not mixed with the sample. This gives thehighest (or reference) signal.

[0174] The invention also contemplates detecting bacterial toxin by a“sandwich” immunoassay method that utilizes antibodies directed againstrecombinant bacterial toxin proteins. Affinity-purified antibodiesdirected against recombinant bacterial toxin proteins are immobilized toa solid support (e.g., microtiter plates). Biological samples suspectedof containing bacterial toxins are then added followed by a washing stepto remove substantially all unbound antitoxin. The biological sample isnext exposed to the reporter substance, which binds to antitoxin and isthen washed free of substantially all unbound reporter substance. Thereporter substance may comprise an antibody with binding specificity forthe antitoxin attached to a molecule which is used to identify thepresence of the reporter substance. Identification of the reportersubstance in the biological tissue indicates the presence of thebacterial toxin.

[0175] It is also contemplated that bacterial toxin be detected bypouring liquids (e.g. soups and other fluid foods and feeds includingnutritional supplements for humans and other animals) over immobilizedantibody which is directed against the bacterial toxin. It iscontemplated that the immobilized antibody will be present in or on suchsupports as cartridges, columns, beads, or any other solid supportmedium. In one embodiment, following the exposure of the liquid to theimmobilized antibody, unbound toxin is substantially removed by washing.The exposure of the liquid is then exposed to a reporter substance whichdetects the presence of bound toxin. In a preferred embodiment thereporter substance is an enzyme, fluorescent dye, or radioactivecompound attached to an antibody which is directed against the toxin(i.e., in a “sandwich” immunoassay). It is also contemplated that thedetection system will be developed as necessary (e.g., the addition ofenzyme substrate in enzyme systems; observation using fluorescent lightfor fluorescent dye systems; and quantitation of radioactivity forradioactive systems).

[0176] Experimental

[0177] The following examples serve to illustrate certain preferredembodiments and aspects of the present invention and are not to beconstrued as limiting the scope thereof.

[0178] In the disclosure which follows, the following abbreviationsapply: ° C. (degrees Centigrade); rpm (revolutions per minute);BBS-Tween (borate buffered saline containing Tween); BSA (bovine serumalbumin); ELISA (enzyme-linked immunosorbent assay); CFA (completeFreund's adjuvant); IFA (incomplete Freund's adjuvant); IgG(immunoglobulin G); IgY (immunoglobulin Y); IM (intramuscular); IP(intraperitoneal); IV (intravenous or intravascular); SC (subcutaneous);H₂O (water); HCl (hydrochloric acid); LD₁₀₀ (lethal dose for 100% ofexperimental animals); aa (amino acid); HPLC (high performance liquidchromatography); kD (kilodaltons); gm (grams); μg (micrograms); mg(milligrams); ng (nanograms); μl (microliters); ml (milliliters); mm(millimeters); nm (nanometers); μm (micrometer); M (molar); mM(millimolar); MW (molecular weight); sec (seconds); min(s)(minute/minutes); hr(s) (hour/hours); MgCl, (magnesium chloride); NaCl(sodium chloride); Na₂CO₃ (sodium carbonate); OD₂₈₀ (optical density at280 nm); OD₆₀₀ (optical density at 600 nm); PAGE (polyacrylamide gelelectrophoresis); PBS [phosphate buffered saline (150 mM NaCl, 10 mMsodium phosphate buffer, pH 7.2)]; PEG (polyethylene glycol); PMSF(phenylmethylsulfonyl fluoride); SDS (sodium dodecyl sulfate); Tris(tris(hydroxymethyl)aminomethane); Ensure® ((Ensure®, Ross Laboratories,Columbus Ohio); Enfamil® (Enfamil®, Mead Johnson); w/v (weight tovolume); v/v (volume to volume); Amicon (Amicon, Inc., Beverly, Mass.);Amresco (Amresco, Inc., Solon, Ohio); ATCC (American Type CultureCollection, Rockville, Md.); BBL (Baltimore Biologics Laboratory, (adivision of Becton Dickinson), Cockeysville, Md.); Becton Dickinson(Becton Dickinson Labware, Lincoln Park, N.J.); BioRad (BioRad,Richmond, Calif.); Biotech (C-C Biotech Corp., Poway, Calif.); CharlesRiver (Charles River Laboratories, Wilmington, Mass.); Cocalico(Cocalico Biologicals Inc., Reamstown, Pa.); CytRx (CytRx Corp.,Norcross, Ga.); Falcon (e.g. Baxter Healthcare Corp., McGaw Park, Ill.and Becton Dickinson); FDA (Federal Food and Drug Administration);Fisher Biotech (Fisher Biotech, Springfield, N.J.); GIBCO (Grand IslandBiologic Company/BRL, Grand Island, N.Y.); Gibco-BRL (Life Technologies,Inc., Gaithersburg, Md.); Harlan Sprague Dawley (Harlan Sprague Dawley,Inc., Madison, Wis.); Mallinckrodt (a division of Baxter HealthcareCorp., McGaw Park, Ill.); Millipore (Millipore Corp., Marlborough,Mass.); New England Biolabs (New England Biolabs, Inc., Beverly, Mass.);Novagen (Novagen, Inc., Madison, Wis.); Pharmacia (Pharmacia, Inc.,Piscataway, N.J.); Qiagen (Qiagen, Chatsworth, Calif.); Sasco (Sasco,Omaha, Nebr.); Showdex (Showa Denko America, Inc., New York, N.Y.);Sigma (Sigma Chemical Co., St. Louis, Mo.); Sterogene (Sterogene, Inc.,Arcadia, Calif.); Tech Lab (Tech Lab, Inc., Blacksburg, Va.); andVaxcell (Vaxcell, Inc., a subsidiary of CytRX Corp., Norcross, Ga.).

[0179] When a recombinant protein is described in the specification itis referred to in a short-hand manner by the amino acids in the toxinsequence present in the recombinant protein rounded to the nearest 10.For example, the recombinant protein pMB1850-2360 contains amino acids1852 through 2362 of the C. difficile toxin B protein. The specificationgives detailed construction details for all recombinant proteins suchthat one skilled in the art will know precisely which amino acids arepresent in a given recombinant protein.

EXAMPLE 1 Production of High-Titer Antibodies to Clostridium difficileOrganisms in a Hen

[0180] Antibodies to certain pathogenic organisms have been shown to beeffective in treating diseases caused by those organisms. It has notbeen shown whether antibodies can be raised, against Clostridiumdifficile, which would be effective in treating infection by thisorganism. Accordingly, C. difficile was tested as immunogen forproduction of hen antibodies.

[0181] To determine the best course for raising high-titer eggantibodies against whole C. difficile organisms, different immunizingstrains and different immunizing concentrations were examined. Theexample involved (a) preparation of the bacterial immunogen, (b)immunization, (c) purification of anti-bacterial chicken antibodies, and(d) detection of anti-bacterial antibodies in the purified IgYpreparations.

[0182] a) Preparation Of Bacterial Immunogen C. difficile strains 43594(serogroup A) and 43596 (serogroup C) were originally obtained from theATCC. These two strains were selected because they represent two of themost commonly-occurring serogroups isolated from patients withantibiotic-associated pseudomembranous colitis. [Delmee et al., J. Clin.Microbiol., 28(10):2210 (1990).] Additionally, both of these strainshave been previously characterized with respect to their virulence inthe Syrian hamster model for C. difficile infection. [Delmee et al., J.Med Microbiol., 33:85 (1990).]

[0183] The bacterial strains were separately cultured on brain heartinfusion agar for 48 hours at 37° C. in a Gas Pack 100 Jar (BBL,Cockeysville, Md.) equipped with a Gas Pack Plus anaerobic envelope(BBL). Forty-eight hour cultures were used because they produce bettergrowth and the organisms have been found to be more cross-reactive withrespect to their surface antigen presentation. The greater the degree ofcross-reactivity of our IgY preparations, the better the probability ofa broad range of activity against different strains/serogroups. [Toma etal., J. Clin. Microbiol., 26(3):426 (1988).]

[0184] The resulting organisms were removed from the agar surface usinga sterile dacron-tip swab, and were suspended in a solution containing0.4% formaldehyde in PBS, pH 7.2. This concentration of formaldehyde hasbeen reported as producing good results for the purpose of preparingwhole-organism immunogen suspensions for the generation of polyclonalanti-C. difficile antisera in rabbits. [Delmee et al., J. Clin.Microbiol., 21:323 (1985); Davies et al., Microbial Path., 9:141(1990).] In this manner, two separate bacterial suspensions wereprepared, one for each strain. The two suspensions were then incubatedat 4° C. for 1 hour. Following this period of formalin-treatment, thesuspensions were centrifuged at 4,200×g for 20 min., and the resultingpellets were washed twice in normal saline. The washed pellets, whichcontained formalin-treated whole organisms, were resuspended in freshnormal saline such that the visual turbidity of each suspensioncorresponded to a #7 McFarland standard. [M. A. C. Edelstein,“Processing Clinical Specimens for Anaerobic Bacteria: Isolation andIdentification Procedures,” in S. M. Finegold et al (eds.)., Bailey andScott's Diagnostic Microbiology, pp. 477-507, C. V. Mosby Co., (1990).The preparation of McFarland nephelometer standards and thecorresponding approximate number of organisms for each tube aredescribed in detail at pp. 172-173 of this volume.] Each of the two #7suspensions was then split into two separate volumes. One volume of eachsuspension was volumetrically adjusted, by the addition of saline, tocorrespond to the visual turbidity of a #1 McFarland standard. [Id.] The#1 suspensions contained approximately 3×10⁸ organisms/ml, and the #7suspensions contained approximately 2×10⁹ organisms/ml. [Id.] The fourresulting concentration-adjusted suspensions of formalin-treated C.difficile organisms were considered to be “bacterial immunogensuspensions.” These suspensions were used immediately after preparationfor the initial immunization. [See section (b).]

[0185] The formalin-treatment procedure did not result in 100%non-viable bacteria in the immunogen suspensions. In order to increasethe level of killing, the formalin concentration and length of treatmentwere both increased for subsequent immunogen preparations, as describedbelow in Table 3. (Although viability was decreased with the strongerformalin treatment, 100% inviability of the bacterial immunogensuspensions was not reached.) Also, in subsequent immunogenpreparations, the formalin solutions were prepared in normal salineinstead of PBS. At day 49, the day of the fifth immunization, the excessvolumes of the four previous bacterial immunogen suspensions were storedfrozen at −70° C. for use during all subsequent immunizations.

[0186] b) Immunization

[0187] For the initial immunization, 1.0 ml volumes of each of the fourbacterial immunogen suspensions described above were separatelyemulsified in 1.2 ml volumes of CFA (GIBCO). For each of the fouremulsified immunogen suspensions, two four-month old White Leghorn henspre-laying) were immunized. (It is not necessary to use pre-laying hens;actively-laying hens can also be utilized.) Each hen received a totalvolume of approximately 1.0 ml of a single emulsified immunogensuspension via four injections (two subcutaneous and two intramuscular)of approximately 250 μl per site. In this manner, a total of fourdifferent immunization combinations, using two hens per combination,were initiated for the purpose of evaluating both the effect ofimmunizing concentration on egg yolk antibody (IgY) production, andinterstrain cross-reactivity of IgY raised against heterologous strains.The four immunization groups are summarized in Table 3. TABLE 3Immunization Groups Approximate Group Designation Immunizing StrainImmunizing Dose CD 43594, #1 C. difficile 1.5 × 10⁸ organisms/hen strain43594 CD 43594, #7 ″ 1.0 × 10⁹ organisms/hen CD 43596, #1 C. difficile1.5 × 10⁸ organisms/hen strain 43596 CD 43596, #7 ″ 1.0 × 10⁹organisms/hen

[0188] The time point for the first series of immunizations wasdesignated as “day zero.” All subsequent immunizations were performed asdescribed above except that the bacterial immunogen suspensions wereemulsified using IFA (GIBCO) instead of CFA, and for the later timepoint immunization, the stored frozen suspensions were used instead offreshly-prepared suspensions. The immunization schedule used is listedin Table 4. TABLE 4 Immunization Schedule Immunogen Day Of ImmunizationFormalin-Treatment Preparation Used 0 1%, 1 hr. freshly-prepared 14 1%,overnight ″ 21 1%, overnight ″ 35 1%, 48 hrs. ″ 49 1%, 72 hrs. ″ 70 ″stored frozen 85 ″ ″ 105 ″ ″

[0189] c) Purification of Anti-Bacterial Chicken Antibodies

[0190] Groups of four eggs were collected per immunization group betweendays 80 and 84 post-initial immunization, and chicken immunoglobulin(IgY) was extracted according to a modification of the procedure of A.Polson et al., Immunol. Comm., 9:495 (1980). A gentle stream ofdistilled water from a squirt bottle was used to separate the yolks fromthe whites, and the yolks were broken by dropping them through a funnelinto a graduated cylinder. The four individual yolks were pooled foreach group. The pooled, broken yolks were blended with 4 volumes of eggextraction buffer to improve antibody yield (egg extraction buffer is0.01 M sodium phosphate, 0.1 M NaCl, pH 7.5, containing 0.005%thimerosal), and PEG 8000 (Amresco) was added to a concentration of3.5%. When all the PEG dissolved, the protein precipitates that formedwere pelleted by centrifugation at 13,000×g for 10 minutes. Thesupernatants were decanted and filtered through cheesecloth to removethe lipid layer, and the PEG was added to the supernatants to a finalconcentration of 12% (the supernatants were assumed to contain 3.5%PEG). After a second centrifugation, the supernatants were discarded andthe pellets were centrifuged a final time to extrude the remaining PEG.These crude IgY pellets were then dissolved in the original yolk volumeof egg extraction buffer and stored at 4° C. As an additional control, apreimmune IgY solution was prepared as described above, using eggscollected from unimmunized hens.

[0191] d) Detection of Anti-Bacterial Antibodies in the Purified IgYPreparations

[0192] In order to evaluate the relative levels of specific anti-C.difficile activity in the IgY preparations described above, a modifiedversion of the whole-organism ELISA procedure of N. V. Padhye et al., J.Clin. Microbiol. 29:99-103 (1990) was used. Frozen organisms of both C.difficile strains described above were thawed and diluted to aconcentration of approximately 1×10⁷ organisms/ml using PBS, pH 7.2. Inthis way, two separate coating suspensions were prepared, one for eachimmunizing strain. Into the wells of 96-well microtiter plates (Falcon,Pro-Bind Assay Plates) were placed 100 μl volumes of the coatingsuspensions. In this manner, each plate well received a total ofapproximately 1×10⁶ organisms of one strain or the other. The plateswere then incubated at 4° C. overnight. The next morning, the coatingsuspensions were decanted, and all wells were washed three times usingPBS. In order to block non-specific binding sites, 100 μl of 0.5% BSA(Sigma) in PBS was then added to each well, and the plates wereincubated for 2 hours at room temperature. The blocking solution wasdecanted, and 100 μl volumes of the IgY preparations described abovewere initially diluted 1:500 with a solution of 0.1% BSA in PBS, andthen serially diluted in 1:5 steps. The following dilutions were placedin the wells: 1:500, 1:2,500, 1:62,5000, 1:312,500, and 1:1,562,500. Theplates were again incubated for 2 hours at room temperature. Followingthis incubation, the IgY-containing solutions were decanted, and thewells were washed three times using BBS-Tween (0.1 M boric acid, 0.025 Msodium borate, 1.0 M NaCl, 0.1% Tween-20), followed by two washes usingPBS-Tween (01% Tween-20), and finally, two washes using PBS only. Toeach well, 100 μl of a 1:750 dilution of rabbit anti-chicken IgG(whole-molecule)-alkaline phosphatase conjugate (Sigma) (diluted in 0.1%BSA in PBS) was added. The plates were again incubated for 2 hours atroom temperature. The conjugate solutions were decanted and the plateswere washed as described above, substituting 50 mM Na₂CO₃, pH 9.5 forthe PBS in the final wash. The plates were developed by the addition of100 μl of a solution containing 1 mg/ml para-nitrophenyl phosphate(Sigma) dissolved in 50 mM Na₂CO₃, 10 mM MgCl₂, pH 9.5 to each well, andincubating the plates at room temperature in the dark for 45 minutes.The absorbance of each well was measured at 410 nm using a Dynatech MR700 plate reader. In this manner, each of the four IgY preparationsdescribed above was tested for reactivity against both of the immunizingC. difficile strains; strain-specific, as well as cross-reactiveactivity was determined.

[0193] Table 5 shows the results of the whole-organism ELISA. All fourIgY preparations demonstrated significant levels of activity, to adilution of 1:62,500 or greater against both of the immunizing organismstrains. Therefore, antibodies raised against one strain were highlycross-reactive with the other strain, and vice versa. The immunizingconcentration of organisms did not have a significant effect onorganism-specific IgY production, as both concentrations producedapproximately equivalent responses. Therefore, the lower immunizingconcentration of approximately 1.5×10⁸ organisms/hen is the preferredimmunizing concentration of the two tested. The preimmune IgYpreparation appeared to possess relatively low levels of C.difficile-reactive activity to a dilution of 1:500, probably due toprior exposure of the animals to environmental clostridia.

[0194] An initial whole-organism ELISA was performed using IgYpreparations made from single CD 43594, #1 and CD 43596, #1 eggscollected around day 50 (data not shown). Specific titers were found tobe 5 to 10-fold lower than those reported in Table 5. These resultsdemonstrate that it is possible to begin immunizing hens prior to thetime that they begin to lay eggs, and to obtain high titer specific IgYfrom the first eggs that are laid. In other words, it is not necessaryto wait for the hens to begin laying before the immunization schedule isstarted. TABLE 5 Results Of The Anti-C. difficile Whole-Organism ELISADilution Of 43594- 43596- IgY Preparation IgY Prep Coated Wells CoatedWells CD 43594, #1 1:500 1.746 1.801 1:2,500 1.092 1.670 1:12,500 0.2020.812 1:62,500 0.136 0.179 1:312,500 0.012 0.080 1:1,562,500 0.002 0.020CD 43594, #7 1:500 1.780 1.771 1:2,500 1.025 1.078 1:12,500 0.188 0.3821:62,500 0.052 0.132 1:312,500 0.022 0.043 1:1,562,500 0.005 0.024 CD43596, #1 1:500 1.526 1.790 1:2,500 0.832 1.477 1:12,500 0.247 0.4521:62,500 0.050 0.242 1:312,500 0.010 0.067 1:1,562,500 0.000 0.036 CD43596, #7 1:500 1.702 1.505 1:2,500 0.706 0.866 1:12,500 0.250 0.2821:62,500 0.039 0.078 1:312,500 0.002 0.017 1:1,562,500 0.000 0.010Preimmune IgY 1:500 0.142 0.309 1:2,500 0.032 0.077 1:12,500 0.006 0.0241:62,500 0.002 0.012 1:312,500 0.004 0.010 1:1,562,500 0.002 0.014

EXAMPLE 2 Treatment Of C. difficile Infection with Anti-C. difficileAntibody

[0195] In order to determine whether the immune IgY antibodies raisedagainst whole C. difficile organisms were capable of inhibiting theinfection of hamsters by C. difficile, hamsters infected by thesebacteria were utilized. [Lyerly et al., Infect. Immun., 59:2215-2218(1991).] This example involved: (a) determination of the lethal dose ofC. difficile organisms; and (b) treatment of infected animals withimmune antibody or control antibody in nutritional solution.

[0196] a) Determination of the Lethal Dose of C. difficile Organisms

[0197] Determination of the lethal dose of C. difficile organisms wascarried out according to the model described by D. M. Lyerly et al.,Infect. Immun., 59:2215-2218 (1991). C. difficile strain ATCC 43596(serogroup C, ATCC) was plated on BHI agar and grown anaerobically (BBLGas Pak 100 system) at 37° C. for 42 hours. Organisms were removed fromthe agar surface using a sterile dacron-tip swab and suspended insterile 0.9% NaCl solution to a density of 10⁸ organisms/ml.

[0198] In order to determine the lethal dose of C. difficile in thepresence of control antibody and nutritional formula, non-immune eggswere obtained from unimmunized hens and a 12% PEG preparation made asdescribed in Example 1(c). This preparation was redissolved in onefourth the original yolk volume of vanilla flavor Ensure®.

[0199] Starting on day one, groups of female Golden Syrian hamsters(Harlan Sprague Dawley), 8-9 weeks old and weighing approximately 100gm, were orally administered 1 ml of the preimmune/Ensure® formula attime zero, 2 hours, 6 hours, and 10 hours. At 1 hour, animals wereorally administered 3.0 mg clindamycin HCl (Sigma) in 1 ml of water.This drug predisposes hamsters to C. difficile infection by altering thenormal intestinal flora. On day two, the animals were given 1 ml of thepreimmune IgY/Ensure® formula at time zero, 2 hours, 6 hours, and 10hours. At 1 hour on day two, different groups of animals were inoculatedorally with saline (control), or 10², 10⁴, 10⁶, or 10⁸ C. difficileorganisms in 1 ml of saline. From days 3-12, animals were given 1 ml ofthe preimmune IgY/Ensure® formula three times daily and observed for theonset of diarrhea and death. Each animal was housed in an individualcage and was offered food and water ad libitum.

[0200] Administration of 10⁶-10⁸ organisms resulted in death in 3-4 dayswhile the lower doses of 10²-10⁴ organisms caused death in 5 days. Cecalswabs taken from dead animals indicated the presence of C. difficile.Given the effectiveness of the 10² dose, this number of organisms waschosen for the following experiment to see if hyperimmune anti-C.difficile antibody could block infection.

[0201] b) Treatment of Infected Animals with Immune Antibody or ControlAntibody in Nutritional Formula

[0202] The experiment in (a) was repeated using three groups of sevenhamsters each. Group A received no clindamycin or C. difficile and wasthe survival control. Group B received clindamycin, 10² C. difficileorganisms and preimmune IgY on the same schedule as the animals in (a)above. Group C received clindamycin, 10² C. difficile organisms, andhyperimmune anti-C. difficile IgY on the same schedule as Group B. Theanti-C. difficile IgY was prepared as described in Example 1 except thatthe 12% PEG preparation was dissolved in one fourth the original yolkvolume of Ensure®.

[0203] All animals were observed for the onset of diarrhea or otherdisease symptoms and death. Each animal was housed in an individual cageand was offered food and water ad libitum. The results are shown inTable 6. TABLE 6 The Effect Of Oral Feeding Of Hyperimmune IgY Antibodyon C. difficile Infection Animal Group Time To Diarrhea^(a) Time ToDeath^(a) A pre-immune IgY only no diarrhea no deaths B Clindamycin, 30hrs. 49 hrs. C. difficile, preimmune IgY C Clindamycin, 33 hrs. 56 hrs.C. difficile, immune IgY

[0204] Hamsters in the control group A did not develop diarrhea andremained healthy during the experimental period. Hamsters in groups Band C developed diarrheal disease. Anti-C. difficile IgY did not protectthe animals from diarrhea or death, all animals succumbed in the sametime interval as the animals treated with preimmune IgY. Thus, whileimmunization with whole organisms apparently can improve sub-lethalsymptoms with particular bacteria (see U.S. Pat. No. 5,080,895 to H.Tokoro), such an approach does not prove to be productive to protectagainst the lethal effects of C. difficile.

EXAMPLE 3 Production of C. botulinum Type A Antitoxin in Hens

[0205] In order to determine whether antibodies could be raised againstthe toxin produced by clostridial pathogens, which would be effective intreating clostridial diseases, antitoxin to C. botulinum type A toxinwas produced. This example involves: (a) toxin modification; (b)immunization; (c) antitoxin collection; (d) antigenicity assessment; and(e) assay of antitoxin titer.

[0206] a) Toxin Modification

[0207]C. botulinum type A toxoid was obtained from B. R. DasGupta. Fromthis, the active type A neurotoxin (M. W. approximately 150 kD) waspurified to greater than 99% purity, according to published methods. [B.R. DasGupta & V. Sathyamoorthy, Toxicon, 22:415 (1984).] The neurotoxinwas detoxified with formaldehyde according to published methods. [B. R.Singh & B. R. DasGupta, Toxicon, 27:403 (1989).]

[0208] b) Immunization

[0209]C. botulinum toxoid for immunization was dissolved in PBS (1mg/ml) and was emulsified with an approximately equal volume of CFA(GIBCO) for initial immunization or IFA for booster immunization. On dayzero, two white leghorn hens, obtained from local breeders, were eachinjected at multiple sites (intramuscular and subcutaneous) with 1 mlinactivated toxoid emulsified in 1 ml CFA. Subsequent boosterimmunizations were made according to the following schedule for day ofinjection and toxoid amount: days 14 and 21-0.5 mg; day 171-0.75 mg;days 394, 401, 409-0.25 mg. One hen received an additional booster of0.150 mg on day 544.

[0210] c) Antitoxin Collection

[0211] Total yolk immunoglobulin (IgY) was extracted as described inExample 1(c) and the IgY pellet was dissolved in the original yolkvolume of PBS with thimerosal.

[0212] d) Antigenicity Assessment

[0213] Eggs were collected from day 409 through day 423 to assesswhether the toxoid was sufficiently immunogenic to raise antibody. Eggsfrom the two hens were pooled and antibody was collected as described inthe standard PEG protocol. [Example 1(c).] Antigenicity of the botulinaltoxin was assessed on Western blots. The 150 kD detoxified type Aneurotoxin and unmodified, toxic, 300 kD botulinal type A complex (toxinused for intragastric route administration for animal gut neutralizationexperiments; see Example 6) were separated on a SDS-polyacrylamidereducing gel. The Western blot technique was performed according to themethod of Towbin. [H. Towbin et al., Proc. Natl. Acad. Sci. USA, 76:4350(1979).] Ten μg samples of C. botulinum complex and toxoid weredissolved in SDS reducing sample buffer (1% SDS, 0.5% 2-mercaptoethanol,50 mM Tris, pH 6.8, 10% glycerol, 0.025% w/v bromphenol blue, 10%β-mercaptoethanol), heated at 95° C. for 10 min and separated on a 1 mmthick 5% SDS-polyacrylamide gel. [K. Weber and M. Osborn, “Proteins andSodium Dodecyl Sulfate: Molecular Weight Determination on PolyacrylamideGels and Related Procedures,” in The Proteins, 3d Edition (H. Neurath &R. L. Hill, eds), pp. 179-223, (Academic Press, NY, 1975).] Part of thegel was cut off and the proteins were stained with Coomassie Blue. Theproteins in the remainder of the gel were transferred to nitrocelluloseusing the Milliblot-SDE electro-blotting system (Millipore) according tomanufacturer's directions. The nitrocellulose was temporarily stainedwith 10% Ponceau S [S. B. Carroll and A. Laughon, “Production andPurification of Polyclonal Antibodies to the Foreign Segment ofβ-galactosidase Fusion Proteins,” in DNA Cloning: A Practical Approach,Vol.III, (D. Glover, ed.), pp. 89-111, IRL Press, Oxford, (1987)] tovisualize the lanes, then destained by running a gentle stream ofdistilled water over the blot for several minutes. The nitrocellulosewas immersed in PBS containing 3% BSA overnight at 4° C. to block anyremaining protein binding sites.

[0214] The blot was cut into strips and each strip was incubated withthe appropriate primary antibody. The avian anti-C. botulinum antibodies[described in (c)] and pre-immune chicken antibody (as control) werediluted 1:125 in PBS containing 1 mg/ml BSA for 2 hours at roomtemperature. The blots were washed with two changes each of largevolumes of PBS, BBS-Tween and PBS, successively (10 min/wash). Goatanti-chicken IgG alkaline phosphatase conjugated secondary antibody(Fisher Biotech) was diluted 1:500 in PBS containing 1 mg/ml BSA andincubated with the blot for 2 hours at room temperature. The blots werewashed with two changes each of large volumes of PBS and BBS-Tween,followed by one change of PBS and 0.1 M Tris-HCl, pH 9.5. Blots weredeveloped in freshly prepared alkaline phosphatase substrate buffer (100μg/ml nitroblue tetrazolium (Sigma), 50 μg/ml 5-bromo-4-chloro-3-indolylphosphate (Sigma), 5 mM MgCl₂ in 50 mM Na₂CO₃, pH 9.5).

[0215] The Western blots are shown in FIG. 1. The anti-C. botulinum IgYreacted to the toxoid to give a broad immunoreactive band at about145-150%D on the reducing gel. This toxoid is refractive to disulfidecleavage by reducing agents due to formalin crosslinking. The immune IgYreacted with the active toxin complex, a 97 kD C. botulinum type A heavychain and a 53 kD light chain. The preimmune IgY was unreactive to theC. botulinum complex or toxoid in the Western blot.

[0216] e) Antitoxin Antibody Titer

[0217] The IgY antibody titer to C. botulinum type A toxoid of eggsharvested between day 409 and 423 was evaluated by ELISA, prepared asfollows. Ninety-six-well Falcon Pro-bind plates were coated overnight at4° C. with 100 μl/well toxoid [B. R. Singh & B. R. Das Gupta, Toxicon27:403 (1989)] at 2.5 μg/ml in PBS, pH 7.5 containing 0.005% thimerosal.The following day the wells were blocked with PBS containing 1% BSA for1 hour at 37° C. The IgY from immune or preimmune eggs was diluted inPBS containing 1% BSA and 0.05% Tween 20 and the plates were incubatedfor 1 hour at 37° C. The plates were washed three times with PBScontaining 0.05% Tween 20 and three times with PBS alone. Alkalinephosphatase-conjugated goat-anti-chicken IgG (Fisher Biotech) wasdiluted 1:750 in PBS containing 1% BSA and 0.05% Tween 20, added to theplates, and incubated 1 hour at 37° C. The plates were washed as before,and p-nitrophenyl phosphate (Sigma) at 1 mg/ml in 0.05 M Na₂CO₃, pH 9.5,10 mM MgCl₂ was added.

[0218] The results are shown in FIG. 2. Chickens immunized with thetoxoid generated high titers of antibody to the immunogen. Importantly,eggs from both immunized hens had significant anti-immunogen antibodytiters as compared to preimmune control eggs. The anti-C. botulinum IgYpossessed significant activity, to a dilution of 1:93,750 or greater.

EXAMPLE 4 Preparation of Avian Egg Yolk Immunoglobulin in an OrallyAdministrable Form

[0219] In order to administer avian IgY antibodies orally toexperimental mice, an effective delivery formula for the IgY had to bedetermined. The concern was that if the crude IgY was dissolved in PBS,the saline in PBS would dehydrate the mice, which might prove harmfulover the duration of the study. Therefore, alternative methods of oraladministration of IgY were tested. The example involved: (a) isolationof immune IgY; (b) solubilization of IgY in water or PBS, includingsubsequent dialysis of the IgY-PBS solution with water to eliminate orreduce the salts (salt and phosphate) in the buffer; and (c) comparisonof the quantity and activity of recovered IgY by absorbance at 280 nmand PAGE, and enzyme-linked immunoassay (ELISA).

[0220] a) Isolation of Immune IgY

[0221] In order to investigate the most effective delivery formula forIgY, we used IgY which was raised against Crotalus durissus terrificusvenom. Three eggs were collected from hens immunized with the C.durissus terrificus venom and IgY was extracted from the yolks using themodified Polson procedure described by Thalley and Carroll[Bio/Technology, 8:934-938 (1990)] as described in Example 1(c).

[0222] The egg yolks were separated from the whites, pooled, and blendedwith four volumes of PBS. Powdered PEG 8000 was added to a concentrationof 3.5%. The mixture was centrifuged at 10,000 rpm for 10 minutes topellet the precipitated protein, and the supernatant was filteredthrough cheesecloth to remove the lipid layer. Powdered PEG 8000 wasadded to the supernatant to bring the final PEG concentration to 12%(assuming a PEG concentration of 3.5% in the supernatant). The 12%.PEG/IgY mixture was divided into two equal volumes and centrifuged topellet the IgY.

[0223] b) Solubilization of the IgY in Water or PBS

[0224] One pellet was resuspended in ½ the original yolk volume of PBS,and the other pellet was resuspended in ½ the original yolk volume ofwater. The pellets were then centrifuged to remove any particles orinsoluble material. The IgY in PBS solution dissolved readily but thefraction resuspended in water remained cloudy.

[0225] In order to satisfy anticipated sterility requirements for orallyadministered antibodies, the antibody solution needs to befilter-sterilized (as an alternative to heat sterilization which woulddestroy the antibodies). The preparation of IgY resuspended in water wastoo cloudy to pass through either a 0.2 or 0.45 μm membrane filter, so10 ml of the PBS resuspended fraction was dialyzed overnight at roomtemperature against 250 ml of water. The following morning the dialysischamber was emptied and refilled with 250 ml of fresh H₂O for a seconddialysis. Thereafter, the yields of soluble antibody were determined atOD₂₈₀ and are compared in Table 7. TABLE 7 Dependence Of IgY Yield OnSolvents Absorbance Of 1:10 Percent Fraction Dilution At 280 nm RecoveryPBS dissolved 1.149 100% H₂O dissolved 0.706 61% PBS dissolved/H₂Odialyzed 0.885 77%

[0226] Resuspending the pellets in PBS followed by dialysis againstwater recovered more antibody than directly resuspending the pellets inwater (77% versus 61%). Equivalent volumes of the IgY preparation in PBSor water were compared by PAGE, and these results were in accordancewith the absorbance values (data not shown).

[0227] c) Activity of IgY Prepared with Different Solvents

[0228] An ELISA was performed to compare the binding activity of the IgYextracted by each procedure described above. C. durissus terrificus(C.d.t.) venom at 2.5 μg/ml in PBS was used to coat each well of a96-well microtiter plate. The remaining protein binding sites wereblocked with PBS containing 5 mg/ml BSA. Primary antibody dilutions (inPBS containing 1 mg/ml BSA) were added in duplicate. After 2 hours ofincubation at room temperature, the unbound primary antibodies wereremoved by washing the wells with PBS, BBS-Tween, and PBS. The speciesspecific secondary antibody (goat anti-chicken immunoglobulinalkaline-phosphatase conjugate (Sigma) was diluted 1:750 in PBScontaining 1 mg/ml BSA and added to each well of the microtiter plate.After 2 hours of incubation at room temperature, the unbound secondaryantibody was removed by washing the plate as before, and freshlyprepared alkaline phosphatase substrate (Sigma) at 1 mg/ml in 50 mMNa₂CO₃, 10 mM MgCl₂, pH 9.5 was added to each well. The colordevelopment was measured on a Dynatech MR 700 microplate reader using a412 nm filter. The results are shown in Table 8.

[0229] The binding assay results parallel the recovery values in Table7, with PBS-dissolved IgY showing slightly more activity than thePBS-dissolved/H₂O dialyzed antibody. The water-dissolved antibody hadconsiderably less binding activity than the other preparations.

EXAMPLE 5 Survival of Antibody Activity After Passage Through theGastrointestinal Tract

[0230] In order to determine the feasibility of oral administration ofantibody, it was of interest to determine whether orally administeredIgY survived passage through the gastrointestinal tract. The exampleinvolved: (a) oral administration of specific immune antibody mixed witha nutritional formula; and (b) assay of antibody activity extracted fromfeces. TABLE 8 Antigen-Binding Activity Of IgY Prepared With DifferentSolvents Dilution Preimmune PBS Dissolved H₂O Dissolved PBS/H₂O 1:5000.005 1.748 1.577 1.742 1:2,500 0.004 0.644 0.349 0.606 1:12,500 0.0010.144 0.054 0.090 1:62,500 0.001 0.025 0.007 0.016 1:312,500 0.010 0.0000.000 0.002

[0231] a) Oral Administration of Antibody

[0232] The IgY preparations used in this example are the samePBS-dissolve 20 dialyzed antivenom materials obtained in Example 4above, mixed with an equal volume of Enfamil®. Two mice were used inthis experiment, each receiving a different diet as follows:

[0233] 1) water and food as usual;

[0234] 2) immune IgY preparation dialyzed against water and mixed 1:1with Enfamil®. (The mice were given the corresponding mixture as theironly source of food and water).

[0235] b) Antibody Activity after Ingestion

[0236] After both mice had ingested their respective fluids, each tubewas refilled with approximately 10 ml of the appropriate fluid firstthing in the morning. By mid-morning there was about 4 to 5 ml of liquidleft in each tube. At this point stool samples were collected from eachmouse, weighed, and dissolved in approximately 500 μl PBS per 100 mgstool sample. One hundred and sixty mg of control stools (no antibody)and 99 mg of experimental stools (specific antibody) in 1.5 ml microfugetubes were dissolved in 800 and 500 μl PBS, respectively. The sampleswere heated at 37° C. for 10 minutes and vortexed vigorously. Theexperimental stools were also broken up with a narrow spatula Eachsample was centrifuged for 5 minutes in a microfuge and thesupernatants, presumably containing the antibody extracts, werecollected. The pellets were saved at 2-8° C. in case future extractswere needed. Because the supernatants were tinted, they were dilutedfive-fold in PBS containing 1 mg/ml BSA for the initial dilution in theenzyme immunoassay (ELISA). The primary extracts were then dilutedfive-fold serially from this initial dilution. The volume of primaryextract added to each well was 190 μl. The ELISA was performed exactlyas described in Example 4. TABLE 9 Specific Antibody Activity AfterPassage Through The Gastrointestinal Tract Dilution Preimmune IgYControl Fecal Extract EXP. Fecal Extract 1:5 <0 0.000 0.032 1:25 0.016<0 0.016 1:125 <0 <0 0.009 1:625 <0 0.003 0.001 1:3125 <0 <0 0.000

[0237] There was some active antibody in the fecal extract from themouse given the specific antibody in Enfamil® formula, but it waspresent at a very low level. Since the samples were assayed at aninitial 1:5 dilution, the binding observed could have been higher withless dilute samples. Consequently, the mice were allowed to continueingesting either regular food and water or the specific IgY in Enfamil®formula, as appropriate, so the assay could be repeated. Another ELISAplate was coated overnight with 5 μg/ml of C.d.t. venom in PBS.

[0238] The following morning the ELISA plate was blocked with 5 mg/mlBSA, and the fecal samples were extracted as before, except that insteadof heating the extracts at 37° C., the samples were kept on ice to limitproteolysis. The samples were assayed undiluted initially, and in 5×serial dilutions thereafter. Otherwise the assay was carried out asbefore. TABLE 10 Specific Antibody Survives Passage Through TheGastrointestinal Tract Dilution Preimmune IgY Control Extract Exp.Extract undiluted 0.003 <0 0.379 1:5 <0 <0 0.071 1:25 0.000 <0 0.0271:125 0.003 <0 0.017 1:625 0.000 <0 0.008 1:3125 0.002 <0 0.002

[0239] The experiment confirmed the previous results, with the antibodyactivity markedly higher. The control fecal extract showed noanti-C.d.t. activity, even undiluted, while the fecal extract from theanti-C.d.t. IgY/Enfamil®-fed mouse showed considerable anti-C.d.t.activity. This experiment (and the previous experiment) clearlydemonstrate that active IgY antibody survives passage through the mousedigestive tract, a finding with favorable implications for the successof IgY antibodies administered orally as a therapeutic or prophylactic.

EXAMPLE 6 In Vivo Neutralization of Type C. botulinum Type A Neurotoxinby Avian Antitoxin Antibody

[0240] This example demonstrated the ability of PEG-purified antitoxin,collected as described in Example 3, to neutralize the lethal effect ofC. botulinum neurotoxin type A in mice. To determine the oral lethaldose (LD₁₀₀) of toxin A, groups of BALB/c mice were given differentdoses of toxin per unit body weight (average body weight of 24 grams).For oral administration, toxin A complex, which contains the neurotoxinassociated with other non-toxin proteins was used. This complex ismarkedly more toxic than purified neurotoxin when given by the oralroute. [I. Ohishi et al., Infect. Immun., 16:106 (1977).] C. botulinumtoxin type A complex, obtained from Eric Johnson (University OfWisconsin, Madison) was 250 μg/ml in 50 mM sodium citrate, pH 5.5,specific toxicity 3×10⁷ mouse LD₅₀/mg with parenteral administration.Approximately 40-50 ng/gm body weight was usually fatal within 48 hoursin mice maintained on conventional food and water. When mice were givena diet ad libitum of only Enfamil® the concentration needed to producelethality was approximately 2.5 times higher (125 ng/gm body weight).Botulinal toxin concentrations of approximately 200 ng/gm body weightwere fatal in mice fed Enfamil® containing preimmune IgY (resuspended inEnfamil® at the original yolk volume).

[0241] The oral LD₁₀₀ of C. botulinum toxin was also determined in micethat received known amounts of a mixture of preimmune IgY-Ensure®delivered orally through feeding needles. Using a 22 gauge feedingneedle, mice were given 250 μl each of a preimmune IgY-Ensure® mixture(preimmune IgY dissolved in ¼ original yolk volume) 1 hour before and ½hour and 5 hours after administering botulinal toxin. Toxinconcentrations given orally ranged from approximately 12 to 312 ng/gmbody weight (0.3 to 7.5 μg per mouse). Botulinal toxin complexconcentration of approximately 40 ng/gm body weight (1 μg per mouse) waslethal in all mice in less than 36 hours.

[0242] Two groups of BALB/c mice, 10 per group, were each given orally asingle dose of 1 μg each of botulinal toxin complex in 100 μl of 50 mMsodium citrate pH 5.5. The mice received 250 μl treatments of a mixtureof either preimmune or immune IgY in Ensure® (¼ original yolk volume) 1hour before and ½ hour, 4 hours, and 8 hours after botulinal toxinadministration. The mice received three treatments per day for two moredays. The mice were observed for 96 hours. The survival and mortalityare shown in Table 11. TABLE 11 Neutralization Of Botulinal Toxin A InVivo Toxin Number Number Dose Antibody Of Mice Of Mice ng/gm Type AliveDead 41.6 non-immune 0 10 41.6 anti-botulinal toxin 10 0

[0243] All mice treated with the preimmune IgY-Ensure® mixture diedwithin 46 hours post-toxin administration. The average time of death inthe mice was 32 hours post toxin administration. Treatments of preimmuneIgY-Ensure® mixture did not continue beyond 24 hours due to extensiveparalysis of the mouth in mice of this group. In contrast, all ten micetreated with the immune anti-botulinal toxin IgY-Ensure® mixturesurvived past 96 hours. Only 4 mice in this group exhibited symptoms ofbotulism toxicity (two mice about 2 days after and two mice 4 days aftertoxin administration). These mice eventually died 5 and 6 days later.Six of the mice in this immune group displayed no adverse effects to thetoxin and remained alive and healthy long term. Thus, the aviananti-botulinal toxin antibody demonstrated very good protection from thelethal effects of the toxin in the experimental mice.

EXAMPLE 7 Production of an Avian Antitoxin Against Clostridium difficileToxin A

[0244] Toxin A is a potent cytotoxin secreted by pathogenic strains ofC. difficile, that plays a direct role in damaging gastrointestinaltissues. In more severe cases of C. difficile intoxication,pseudomembranous colitis can develop which may be fatal. This would beprevented by neutralizing the effects of this toxin in thegastrointestinal tract. As a first step, antibodies were producedagainst a portion of the toxin. The example involved: (a) conjugation ofa synthetic peptide of toxin A to bovine serum albumin; (b) immunizationof hens with the peptide-BSA conjugate; and (c) detection of antitoxinpeptide antibodies by ELISA.

[0245] a) Conjugation Of A Synthetic Peptide Of Toxin A To Bovine SerumAlbumin

[0246] The synthetic peptide CQTIDGKKYYFN—NH₂ (SEQ ID NO:82) wasprepared commercially (Multiple Peptide Systems, San Diego, Calif.) andvalidated to be>80% pure by high-pressure liquid chromatography. Theeleven amino acids following the cysteine residue represent a consensussequence of a repeated amino acid sequence found in Toxin A. [Wren etal., Infect. Immun., 59:3151-3155 (1991).] The cysteine was added tofacilitate conjugation to carrier protein.

[0247] In order to prepare the carrier for conjugation, BSA (Sigma) wasdissolved in 0.01 M NaPO₄, pH 7.0 to a final concentration of 20 mg/mland n-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Pierce) wasdissolved in N,N-dimethyl formamide to a concentration of 5 mg/ml. MBSsolution, 0.51 ml, was added to 3.25 ml of the BSA solution andincubated for 30 minutes at room temperature with stirring every 5minutes. The MBS-activated BSA was then purified by chromatography on aBio-Gel P-10 column (Bio-Rad; 40 ml bed volume) equilibrated with 50 mMNaPO₄, pH 7.0 buffer. Peak fractions were pooled (6.0 ml).

[0248] Lyophilized toxin A peptide (20 mg) was added to the activatedBSA mixture, stirred until the peptide dissolved and incubated 3 hoursat room temperature. Within 20 minutes, the reaction mixture becamecloudy and precipitates formed. After 3 hours, the reaction mixture wascentrifuged at 10,000×g for 10 min and the supernatant analyzed forprotein content. No significant protein could be detected at 280 nm. Theconjugate precipitate was washed three times with PBS and stored at 4°C. A second conjugation was performed with 15 mg of activated BSA and 5mg of peptide and the conjugates pooled and suspended at a peptideconcentration of 10 mg/ml in 10 mM NaPO₄, pH 7.2.

[0249] b) Immunization of Hens with Peptide Conjugate

[0250] Two hens were each initially immunized on day zero by injectioninto two subcutaneous and two intramuscular sites with 1 mg of peptideconjugate that was emulsified in CFA (GIBCO). The hens were boosted onday 14 and day 21 with 1 mg of peptide conjugate emulsified in IFA(GIBCO).

[0251] c) Detection of Antitoxin Peptide Antibodies by ELISA

[0252] IgY was purified from two eggs obtained before immunization(pre-immune) and two eggs obtained 31 and 32 days after the initialimmunization using PEG fractionation as described in Example 1.

[0253] Wells of a 96-well microtiter plate (Falcon Pro-Bind Assay Plate)were coated overnight at 4° C. with 100 μg/ml solution of the toxin Asynthetic peptide in PBS, pH 7.2 prepared by dissolving 1 mg of thepeptide in 1.0 ml of H₂O and dilution of PBS. The pre-immune and immuneIgY preparations were diluted in a five-fold series in a buffercontaining 1% PEG 8000 and 0.1% Tween-20 (v/v) in PBS, pH 7.2. The wellswere blocked for 2 hours at room temperature with 150 μl of a solutioncontaining 5% (v/v) Carnation® nonfat dry milk and 1% PEG 8000 in PBS,pH 7.2. After incubation for 2 hours at room temperature, the wells werewashed, secondary rabbit anti-chicken IgG-alkaline phosphatase (1:750)added, the wells washed again and the color development obtained asdescribed in Example 1. The results are shown in Table 12. TABLE 12Reactivity Of IgY With Toxin Peptide Absorbance At 410 nm Dilution OfPEG Prep Preimmune Immune Anti-Peptide. 1:100  0.013 0.253 1:500  0.0040.039 1:2500 0.004 0.005

[0254] Clearly, the immune antibodies contain titers against thisrepeated epitope of toxin A.

EXAMPLE 8 Production of Avian Antitoxins Against Clostridium difficileNative Toxins A and B

[0255] To determine whether avian antibodies are effective for theneutralization of C. difficile toxins, hens were immunized using nativeC. difficile toxins A and B. The resulting egg yolk antibodies were thenextracted and assessed for their ability to neutralize toxins A and B invitro. The Example involved (a) preparation of the toxin immunogens, (b)immunization, (c) purification of the antitoxins, and (d) assay of toxinneutralization activity.

[0256] a) Preparation of the Toxin Immunogens

[0257] Both C. difficile native toxins A and B, and C. difficiletoxoids, prepared by the treatment of the native toxins withformaldehyde, were employed as immunogens. C. difficile toxoids A and Bwere prepared by a procedure which was modified from published methods(Ehrich et al., Infect. Immun. 28:1041 (1980). Separate solutions (inPBS) of native C. difficile toxin A and toxin B (Tech Lab) were eachadjusted to a concentration of 0.20 mg/ml, and formaldehyde was added toa final concentration of 0.4%. The toxin/formaldehyde solutions werethen incubated at 37° C. for 40 hrs. Free formaldehyde was then removedfrom the resulting toxoid solutions by dialysis against PBS at 4° C. Inpreviously published reports, this dialysis step was not performed.Therefore, free formaldehyde must have been present in their toxoidpreparations. The toxoid solutions were concentrated, using a Centriprepconcentrator unit (Amicon), to a final toxoid concentration of 4.0mg/ml. The two resulting preparations were designated as toxoid A andtoxoid B.

[0258]C. difficile native toxins were prepared by concentrating stocksolutions of toxin A and toxin B (Tech Lab, Inc), using Centriprepconcentrator units (Amicon), to a final concentration of 4.0 mg/ml.

[0259] b) Immunization

[0260] The first two immunizations were performed using the toxoid A andtoxoid B immunogens described above. A total of 3 different immunizationcombinations were employed. For the first immunization group, 0.2 ml oftoxoid A was emulsified in an equal volume of Titer Max adjuvant(CytRx). Titer Max was used in order to conserve the amount of immunogenused, and to simplify the immunization procedure. This immunizationgroup was designated “CTA.” For the second immunization group, 0.1 ml oftoxoid B was emulsified in an equal volume of Titer Max adjuvant. Thisgroup was designated “CTB.” For the third immunization group, 0.2 ml oftoxoid A was first mixed with 0.2 ml of toxoid B, and the resultingmixture was emulsified in 0.4 ml of Titer Max adjuvant. This group wasdesignated “CTAB.” In this way, three separate immunogen emulsions wereprepared, with each emulsion containing a final concentration of 2.0mg/ml of toxoid A (CTA) or toxoid B (CTB) or a mixture of 2.0 mg/mltoxoid A and 2.0 mg/ml toxoid B (CTAB).

[0261] On day 0, White Leghorn hens, obtained from a local breeder, wereimmunized as follows: Group CTA. Four hens were immunized, with each henreceiving 200%g of toxoid A, via two intramuscular (I.M.) injections of50 μl of CTA emulsion in the breast area. Group CTB. One hen wasimmunized with 200 μg of toxoid B, via two I.M. injections of 50 μl ofCTB emulsion in the breast area. Group CTAB. Four hens were immunized,with each hen receiving a mixture containing 200 μg of toxoid A and 200μg of toxoid B, via two I.M. injections of 100 μl of CTAB emulsion inthe breast area. The second immunization was performed 5 weeks later, onday 35, exactly as described for the first immunization above.

[0262] In order to determine whether hens previously immunized with C.difficile toxoids could tolerate subsequent booster immunizations usingnative toxins, a single hen from group CTAB was immunized for a thirdtime, this time using a mixture of the native toxin A and native toxin Bdescribed in section (a) above (these toxins were notformaldehyde-treated, and were used in their active form). This was donein order to increase the amount (titer) and affinity of specificantitoxin antibody produced by the hen over that achieved by immunizingwith toxoids only. On day 62, 0.1 ml of a toxin mixture was preparedwhich contained 200 μg of native toxin A and 200%g of native toxin B.This toxin mixture was then emulsified in 0.1 ml of Titer Max adjuvant.A single CTAB hen was then immunized with the resulting immunogenemulsion, via two I.M. injections of 100 μl each, into the breast area.This hen was marked with a wing band, and observed for adverse effectsfor a period of approximately 1 week, after which time the hen appearedto be in good health.

[0263] Because the CTAB hen described above tolerated the boosterimmunization with native toxins A and B with no adverse effects, it wasdecided to boost the remaining hens with native toxin as well. On day70, booster immunizations were performed as follows: Group CTA. A 0.2 mlvolume of the 4 mg/ml native toxin A solution was emulsified in an equalvolume of Titer Max adjuvant. Each of the 4 hens was then immunized with200 μg of native toxin A, as described for the toxoid A immunizationsabove. Group CTB. A 50 μl volume of the 4 mg/ml native toxin B solutionwas emulsified in an equal volume of Titer Max adjuvant. The hen wasthen immunized with 200 μg of native toxin B, as described for thetoxoid B immunizations above. Group CTAB. A 0.15 ml volume of the 4mg/ml native toxin A solution was first mixed with a 0.15 ml volume the4 mg/ml native toxin B solution. The resulting toxin mixture was thenemulsified in 0.3 ml of Titer Max adjuvant. The 3 remaining hens (thehen with the wing band was not immunized this time) were then immunizedwith 200%g of native toxin A and 200%g of native toxin B as describedfor the toxoid A+ toxoid B immunizations (CTAB) above. On day 85, allhens received a second booster immunization using native toxins, doneexactly as described for the first boost with native toxins above.

[0264] All hens tolerated both booster immunizations with native toxinswith no adverse effects. As previous literature references describe theuse of formaldehyde-treated toxoids, this is apparently the first timethat any immunizations have been performed using native C. difficiletoxins.

[0265] c) Purification of Antitoxins

[0266] Eggs were collected from the hen in group CTB 10-12 daysfollowing the second immunization with toxoid (day 35 immunizationdescribed in section (b) above), and from the hens in groups CTA andCTAB 20-21 days following the second immunization with toxoid. To beused as a pre-immune (negative) control, eggs were also collected fromunimmunized hens from the same flock. Egg yolk immunoglobulin (IgY) wasextracted from the 4 groups of eggs as described in Example 1(c), andthe final IgY pellets were solubilized in the original yolk volume ofPBS without thimerosal. Importantly, thimerosal was excluded because itwould have been toxic to the CHO cells used in the toxin neutralizationassays described in section (d) below.

[0267] d) Assay of Toxin Neutralization Activity

[0268] The toxin neutralization activity of the IgY solutions preparedin section (c) above was determined using an assay system that wasmodified from published methods. [Ehrich et al., Infect. Immun.28:1041-1043 (1992); and McGee et al. Microb. Path. 12:333-341 (1992).]As additional controls, affinity-purified goat anti-C. difficile toxin A(Tech Lab) and affinity-purified goat anti-C. difficile toxin B (TechLab) were also assayed for toxin neutralization activity.

[0269] The IgY solutions and goat antibodies were serially diluted usingF 12 medium (GIBCO) which was supplemented with 2% FCS (GIBCO)(thissolution will be referred to as “medium” for the remainder of thisExample). The resulting antibody solutions were then mixed with astandardized concentration of either native C. difficile toxin A (TechLab), or native C. difficile toxin B (Tech Lab), at the concentrationsindicated below. Following incubation at 37° C. for 60 min., 100 μlvolumes of the toxin+antibody mixtures were added to the wells of96-well microtiter plates (Falcon Microtest III) which contained 2.5×10⁴Chinese Hamster Ovary (CHO) cells per well (the CHO cells were plated onthe previous day to allow them to adhere to the plate wells). The finalconcentration of toxin, or dilution of antibody indicated below refersto the final test concentration of each reagent present in therespective microtiter plate wells. Toxin reference wells were preparedwhich contained CHO cells and toxin A or toxin B at the sameconcentration used for the toxin plus antibody mixtures (these wellscontained no antibody). Separate control wells were also prepared whichcontained CHO cells and medium only. The assay plates were thenincubated for 18-24 hrs. in a 37° C., humidified, 5% CO₂ incubator. Onthe following day, the remaining adherent (viable) cells in the platewells were stained using 0.2% crystal violet (Mallinckrodt) dissolved in2% ethanol, for 10 min. Excess stain was then removed by rinsing withwater, and the stained cells were solubilized by adding 1001 μl of 1%SDS (dissolved in water) to each well. The absorbance of each well wasthen measured at 570 nm, and the percent cytotoxicity of each testsample or mixture was calculated using the following formula:${\% \quad {CHO}\quad {Cell}\quad {Cytotoxicity}} = {\left\lbrack {1 - \left( \frac{{Abs}.\quad {Sample}}{{Abs}.\quad {Control}} \right)} \right\rbrack \times 100}$

[0270] Unlike previous reports which quantitate results visually bycounting cell rounding by microscopy, this Example utilizedspectrophotometric methods to quantitate the C. difficile toxinbioassay. In order to determine the toxin A neutralizing activity of theCTA, CTAB, and pre-immune IgY preparations, as well as theaffinity-purified goat antitoxin A control, dilutions of theseantibodies were reacted against a 0.1 μg/ml concentration of nativetoxin A (this is the approx. 50% cytotoxic dose of toxin A in this assaysystem). The results are shown in FIG. 3.

[0271] Complete neutralization of toxin A occurred with the CTA IgY(antitoxin A, above) at dilutions of 1:80 and lower, while significantneutralization occurred out to the 1:320 dilution. The CTAB IgY(antitoxin A+ toxin B, above) demonstrated complete neutralization atthe 1:320-1:160 and lower dilutions, and significant neutralizationoccurred out to the 1:1280 dilution. The commercially availableaffinity-purified goat antitoxin A did not completely neutralize toxin Aat any of the dilutions tested, but demonstrated significantneutralization out to a dilution of 1:1,280. The preimmune IgY did notshow any toxin A neutralizing activity at any of the concentrationstested. These results demonstrate that IgY purified from eggs laid byhens immunized with toxin A alone, or simultaneously with toxin A andtoxin B, is an effective toxin A antitoxin.

[0272] The toxin B neutralizing activity of the CTAB and pre-immune IgYpreparations, and also the affinity-purified goat antitoxin B controlwas determined by reacting dilutions of these antibodies against aconcentration of native toxin B of 0.1 ng/ml (approximately the 50%cytotoxic dose of toxin B in the assay system). The results are shown inFIG. 4.

[0273] Complete neutralization of toxin B occurred with the CTAB IgY(antitoxin A+toxin B, above) at the 1:40 and lower dilutions, andsignificant neutralization occurred out to the 1:320 dilution. Theaffinity-purified goat antitoxin B demonstrated complete neutralizationat dilutions of 1:640 and lower, and significant neutralization occurredout to a dilution of 1:2,560. The preimmune IgY did not show any toxin Bneutralizing activity at any of the concentrations tested. These resultsdemonstrate that IgY purified from eggs laid by hens immunizedsimultaneously with toxin A and toxin B is an effective toxin Bantitoxin.

[0274] In a separate study, the toxin B neutralizing activity of CTB,CTAB, and pre-immune IgY preparations was determined by reactingdilutions of these antibodies against a native toxin B concentration of0.1 μg/ml (approximately 100% cytotoxic dose of toxin B in this assaysystem). The results are shown in FIG. 5.

[0275] Significant neutralization of toxin B occurred with the CTB IgY(antitoxin B, above) at dilutions of 1:80 and lower, while the CTAB IgY(antitoxin A+toxin B, above) was found to have significant neutralizingactivity at dilutions of 1:40 and lower. The preimmune IgY did not showany toxin B neutralizing activity at any of the concentrations tested.These results demonstrate that IgY purified from eggs laid by hensimmunized with toxin B alone, or simultaneously with toxin A and toxinB, is an effective toxin B antitoxin.

EXAMPLE 9 In Vivo Protection of Golden Syrian Hamsters from C. difficileDisease by Avian Antitoxins Against C. difficile Toxins A and B

[0276] The most extensively used animal model to study C. difficiledisease is the hamster. [Lyerly et al, Infect. Immun. 47:349-352(1992).] Several other animal models for antibiotic-induced diarrheaexist, but none mimic the human form of the disease as closely as thehamster model. [R. Fekety, “Animal Models of Antibiotic-InducedColitis,” in O. Zak and M. Sande (eds.), Experimental Models inAntimicrobial Chemotherapy, Vol. 2, pp.61-72, (1986).] In this model,the animals are first predisposed to the disease by the oraladministration of an antibiotic, such as clindamycin, which alters thepopulation of normally-occurring gastrointestinal flora (Fekety, at61-72). Following the oral challenge of these animals with viable C.difficile organisms, the hamsters develop cecitis, and hemorrhage,ulceration, and inflammation are evident in the intestinal mucosa.[Lyerly et al., Infect. Immun. 47:349-352 (1985).] The animals becomelethargic, develop severe diarrhea, and a high percentage of them diefrom the disease. [Lyerly et al., Infect. Immun. 47:349-352 (1985).]This model is therefore ideally suited for the evaluation of therapeuticagents designed for the treatment or prophylaxis of C. difficiledisease.

[0277] The ability of the avian C. difficile antitoxins, described inExample 1 above, to protect hamsters from C. difficile disease wasevaluated using the Golden Syrian hamster model of C. difficileinfection. The Example involved (a) preparation of the avian C.difficile antitoxins, (b) in vivo protection of hamsters from C.difficile disease by treatment with avian antitoxins, and (c) long-termsurvival of treated hamsters.

[0278] a) Preparation of the Avian C. difficile Antitoxins

[0279] Eggs were collected from hens in groups CTA and CTAB described inExample 1(b) above. To be used as a pre-immune (negative) control, eggswere also purchased from a local supermarket. Egg yolk immunoglobulin(IgY) was extracted from the 3 groups of eggs as described in Example1(c), and the final IgY pellets were solubilized in one fourth theoriginal yolk volume of Ensure® nutritional formula.

[0280] b) In Vivo Protection of Hamsters Against C. difficile Disease byTreatment with Avian Antitoxins

[0281] The avian C. difficile antitoxins prepared in section (a) abovewere evaluated for their ability to protect hamsters from C. difficiledisease using an animal model system which was modified from publishedprocedures. [Fekety, at 61-72; Borriello et al., J. Med. Microbiol.,24:53-64 (1987); Kim et al., Infect. Immun., 55:2984-2992 (1987);Borriello et al., J. Med. Microbiol., 25:191-196 (1988); Delmee andAvesani, J. Med. Microbiol., 33:85-90 (1990); and Lyerly et al., Infect.Immun. 59:2215-2218 (1991).] For the study, three separate experimentalgroups were used, with each group consisting of 7 female Golden Syrianhamsters (Charles River), approximately 10 weeks old and weighingapproximately 100 gms. each. The three groups were designated “CTA,”“CTAB” and “Pre-immune.” These designations corresponded to theantitoxin preparations with which the animals in each group weretreated. Each animal was housed in an individual cage, and was offeredfood and water ad libitum through the entire length of the study. On day1, each animal was orally administered 1.0 ml of one of the threeantitoxin preparations (prepared in section (a) above) at the followingtimepoints: 0 hrs., 4 hrs., and 8 hrs. On day 2, the day 1 treatment wasrepeated. On day 3, at the 0 hr. timepoint, each animal was againadministered antitoxin, as described above. At 1 hr., each animal wasorally administered 3.0 mg of clindamycin-HCl (Sigma) in 1 ml of water.This treatment predisposed the animals to infection with C. difficile.As a control for possible endogenous C. difficile colonization, anadditional animal from the same shipment (untreated) was alsoadministered 3.0 mg of clindamycin-HCl in the same manner. Thisclindamycin control animal was left untreated (and uninfected) for theremainder of the study. At the 4 hr. and 8 hr. timepoints, the animalswere administered antitoxin as described above. On day 4, at the 0 hr.timepoint, each animal was again administered antitoxin as describedabove. At 1 hr., each animal was orally challenged with 1 ml of C.difficile inoculum, which contained approx. 100 C. difficile strain43596 organisms in sterile saline. C. difficile strain 43596, which is aserogroup C strain, was chosen because it is representative of one ofthe most frequently-occurring serogroups isolated from patients withantibiotic-associated pseudomembranous colitis. [Delmee et al., J. Clin.Microbiol., 28:2210-2214 (1985).] In addition, this strain has beenpreviously demonstrated to be virulent in the hamster model ofinfection. [Delmee and Avesani, J. Med. Microbiol., 33:85-90 (1990).] Atthe 4 hr. and 8 hr. timepoints, the animals were administered antitoxinas described above. On days 5 through 13, the animals were administeredantitoxin 3× per day as described for day 1 above, and observed for theonset of diarrhea and death. On the morning of day 14, the final resultsof the study were tabulated. These results are shown in Table 13.

[0282] Representative animals from those that died in the Pre-Immune andCTA groups were necropsied. Viable C. difficile organisms were culturedfrom the ceca of these animals, and the gross pathology of thegastrointestinal tracts of these animals was consistent with thatexpected for C. difficile disease (inflamed, distended, hemorrhagiccecum, filled with watery diarrhea-like material). In addition, theclindamycin control animal remained healthy throughout the entire studyperiod, therefore indicating that the hamsters used in the study had notpreviously been colonized with endogenous C. difficile organisms priorto the start of the study. Following the final antitoxin treatment onday 13, a single surviving animal from the CTA group, and also from theCTAB group, was sacrificed and necropsied. No pathology was noted ineither animal. TABLE 13 Treatment Results No. Animals No. AnimalsTreatment Group Surviving Dead Pre-Immune 1 6 CTA (Antitoxin A only) 5 2CTAB (Antitoxin A + Antitoxin B) 7 0

[0283] Treatment of hamsters with orally-administered toxin A and toxinB antitoxin (group CTAB) successfully protected 7 out of 7 (100%) of theanimals from C. difficile disease. Treatment of hamsters withorally-administered toxin A antitoxin (group CTA) protected 5 out of 7(71%) of these animals from C. difficile disease. Treatment usingpre-immune IgY was not protective against C. difficile disease, as only1 out of 7 (14%) of these animals survived. These results demonstratethat the avian toxin A antitoxin and the avian toxin A+toxin B antitoxineffectively protected the hamsters from C. difficile disease. Theseresults also suggest that although the neutralization of toxin A aloneconfers some degree of protection against C. difficile disease, in orderto achieve maximal protection, simultaneous antitoxin A and antitoxin Bactivity is necessary.

[0284] c) Long-Term Survival of Treated Hamsters

[0285] It has been previously reported in the literature that hamsterstreated with orally-administered bovine antitoxin IgG concentrate areprotected from C. difficile disease as long as the treatment iscontinued, but when the treatment is stopped, the animals developdiarrhea and subsequently die within 72 hrs. [Lyerly et al., Infect.Immun., 59(6):2215-2218 (1991).]

[0286] In order to determine whether treatment of C. difficile diseaseusing avian antitoxins promotes long-term survival following thediscontinuation of treatment, the 4 surviving animals in group CTA, andthe 6 surviving animals in group CTAB were observed for a period of 11days (264 hrs.) following the discontinuation of antitoxin treatmentdescribed in section (b) above. All hamsters remained healthy throughthe entire post-treatment period. This result demonstrates that not onlydoes treatment with avian antitoxin protect against the onset of C.difficile disease (i.e., it is effective as a prophylactic), it alsopromotes long-term survival beyond the treatment period, and thusprovides a lasting cure.

EXAMPLE 10 In Vivo Treatment of Established C. difficile Infection inGolden Syrian Hamsters with Avian Antitoxins Against C. difficile ToxinsA and B

[0287] The ability of the avian C. difficile antitoxins, described inExample 8 above, to treat an established C. difficile infection wasevaluated using the Golden Syrian hamster model. The Example involved(a) preparation of the avian C. difficile antitoxins, (b) in vivotreatment of hamsters with established C. difficile infection, and (c)histologic evaluation of cecal tissue.

[0288] a) Preparation Of The Avian C. difficile Antitoxins

[0289] Eggs were collected from hens in group CTAB described in Example8(b) above, which were immunized with C. difficile toxoids and nativetoxins A and B. Eggs purchased from a local supermarket were used as apre-immune (negative) control. Egg yolk immunoglobulin (IgY) wasextracted from the 2 groups of eggs as described in Example 1(c), andthe final IgY pellets were solubilized in one-fourth the original yolkvolume of Ensure® nutritional formula.

[0290] b) In Vivo Treatment of Hamsters with Established C. difficileInfection

[0291] The avian C. difficile antitoxins prepared in section (a) abovewere evaluated for the ability to treat established C. difficileinfection in hamsters using an animal model system which was modifiedfrom the procedure which was described for the hamster protection studyin Example 8(b) above.

[0292] For the study, four separate experimental groups were used, witheach group consisting of 7 female Golden Syrian hamsters (CharlesRiver), approx. 10 weeks old, weighing approximately 100 gms. each. Eachanimal was housed separately, and was offered food and water ad libitumthrough the entire length of the study.

[0293] On day 1 of the study, the animals in all four groups were eachpredisposed to C. difficile infection by the oral administration of 3.0mg of clindamycin-HCl (Sigma) in 1 ml of water.

[0294] On day 2, each animal in all four groups was orally challengedwith 1 ml of C. difficile inoculum, which contained approximately 100 C.difficile strain 43596 organisms in sterile saline. C. difficile strain43596 was chosen because it is representative of one of the mostfrequently-occurring serogroups isolated from patients withantibiotic-associated pseudomembranous colitis. [Delmee et al., J. Clin.Microbiol., 28:2210-2214 (1990).] In addition, as this was the same C.difficile strain used in all of the previous Examples above, it wasagain used in order to provide experimental continuity.

[0295] On day 3 of the study (24 hrs. post-infection), treatment wasstarted for two of the four groups of animals. Each animal of one groupwas orally administered 1.0 ml of the CTAB IgY preparation (prepared insection (a) above) at the following timepoints: 0 hrs., 4 hrs., and 8hrs. The animals in this group were designated “CTAB-24.” The animals inthe second group were each orally administered 1.0 ml of the pre-immuneIgY preparation (also prepared in section (a) above) at the sametimepoints as for the CTAB group. These animals were designated“Pre-24.” Nothing was done to the remaining two groups of animals on day3.

[0296] On day 4, 48 hrs. post-infection, the treatment described for day3 above was repeated for the CTAB-24 and Pre-24 groups, and wasinitiated for the remaining two groups at the same timepoints. The finaltwo groups of animals were designated “CTAB-48” and “Pre-48”respectively.

[0297] On days 5 through 9, the animals in all four groups wereadministered antitoxin or pre-immune IgY, 3× per day, as described forday 4 above. The four experimental groups are summarized in Table 14.TABLE 14 Experimental Treatment Groups Group Designation ExperimentalTreatment CTAB-24 Infected, treatment w/antitoxin IgY started @ 24 hrs.post-infection. Pre-24 Infected, treatment w/pre-immune IgY started @ 24hrs. post-infection. CTAB-48 Infected, treatment w/antitoxin IgY started@ 48 hrs. post-infection. Pre-48 Infected, treatment w/pre-immune IgYstarted @ 48 hrs. post-infection.

[0298] All animals were observed for the onset of diarrhea and deaththrough the conclusion of the study on the morning of day 10. Theresults of this study are displayed in Table 15. TABLE 15 ExperimentalOutcome-Day 10 Treatment Group No. Animals Surviving No. Animals DeadCTAB-24 6 1 Pre-24 0 7 CTAB-48 4 3 Pre-48 2 5

[0299] Eighty-six percent of the animals which began receiving treatmentwith antitoxin IgY at 24 hrs. post-infection (CTAB-24 above) survived,while 57% of the animals treated with antitoxin IgY starting 48 hrs.post-infection (CTAB-48 above) survived. In contrast, none of theanimals receiving pre-immune IgY starting 24 hrs. post-infection (Pre-24above) survived, and only 29% of the animals which began receivingtreatment with pre-immune IgY at 48 hrs. post-infection (Pre-48 above)survived through the conclusion of the study. These results demonstratethat avian antitoxins raised against C. difficile toxins A and B arecapable of successfully treating established C. difficile infections invivo.

[0300] c) Histologic Evaluation of Cecal Tissue

[0301] In order to further evaluate the ability of the IgY preparationstested in this study to treat established C. difficile infection,histologic evaluations were performed on cecal tissue specimens obtainedfrom representative animals from the study described in section (b)above.

[0302] Immediately following death, cecal tissue specimens were removedfrom animals which died in the Pre-24 and Pre-48 groups. Following thecompletion of the study, a representative surviving animal wassacrificed and cecal tissue specimens were removed from the CTAB-24 andCTAB-48 groups. A single untreated animal from the same shipment asthose used in the study was also sacrificed and a cecal tissue specimenwas removed as a normal control. All tissue specimens were fixedovernight at 4° C. in 10% buffered formalin. The fixed tissues wereparaffin-embedded, sectioned, and mounted on glass microscope slides.The tissue sections were then stained using hematoxylin and eosin (H andE stain), and were examined by light microscopy.

[0303] Upon examination, the tissues obtained from the CTAB-24 andCTAB-48 animals showed no pathology, and were indistinguishable from thenormal control. This observation provides further evidence for theability of avian antitoxins raised against C. difficile toxins A and Bto effectively treat established C. difficile infection, and to preventthe pathologic consequences which normally occur as a result of C.difficile disease.

[0304] In contrast, characteristic substantial mucosal damage anddestruction was observed in the tissues of the animals from the Pre-24and Pre-48 groups which died from C. difficile disease. Normal tissuearchitecture was obliterated in these two preparations, as most of themucosal layer was observed to have sloughed away, and there werenumerous large hemorrhagic areas containing massive numbers oferythrocytes.

EXAMPLE 11 Cloning and Expression of C. difficile Toxin A Fragments

[0305] The toxin A gene has been cloned and sequenced, and shown toencode a protein of predicted MW of 308 kd. [Dove et al., Infect.Immun., 58:480-488 (1990).] Given the expense and difficulty ofisolating native toxin A protein, it would be advantageous to use simpleand inexpensive procaryotic expression systems to produce and purifyhigh levels of recombinant toxin A protein for immunization purposes.Ideally, the isolated recombinant protein would be soluble in order topreserve native antigenicity, since solubilized inclusion body proteinsoften do not fold into native conformations. To allow ease ofpurification, the recombinant protein should be expressed to levelsgreater than 1 mg/liter of E. coli culture.

[0306] To determine whether high levels of recombinant toxin A proteincan be produced in E coli, fragments of the toxin A gene were clonedinto various prokaryotic expression vectors, and assessed for theability to express recombinant toxin A protein in E. coli. Threeprokaryotic expression systems were utilized. These systems were chosenbecause they drive expression of either fusion (pMALc and pGEX2T) ornative (pET23a-c) protein to high levels in E. coli, and allow affinitypurification of the expressed protein on a ligand containing column.Fusion proteins expressed from pGEX vectors bind glutathione agarosebeads, and are eluted with reduced glutathione. pMAL fusion proteinsbind amylose resin, and are eluted with maltose. A poly-histidine tag ispresent at either the N-terminal (pET16b) or C-terminal (pET23a-c) endof pET fusion proteins. This sequence specifically binds Ni₂ ⁺ chelatecolumns, and is eluted with imidazole salts. Extensive descriptions ofthese vectors are available [Williams et al. (1995) DNA Cloning 2:Expression Systems, Glover and Hames, eds. IRL Press, Oxford, pp.15-58], and will not be discussed in detail here. The Example involved(a) cloning of the toxin A gene, (b) expression of large fragments oftoxin A in various prokaryotic expression systems, (c) identification ofsmaller toxin A gene fragments that express efficiently in E. coli, (d)purification of recombinant toxin A protein by affinity chromatography,and (e) demonstration of functional activity of a recombinant fragmentof the toxin A gene. a) Cloning of the Toxin A Gene

[0307] A restriction map of the toxin A gene is shown in FIG. 6. Theencoded protein contains a carboxy terminal ligand binding region,containing multiple repeats of a carbohydrate binding domain. [vonEichel-Streiber and Sauerbom, Gene 96:107-113 (1990).] The toxin A genewas cloned in three pieces, by using either the polymerase chainreaction (PCR) to amplify specific regions, (regions 1 and 2, FIG. 6) orby screening a constructed genomic library for a specific toxin A genefragment (region 3, FIG. 6). The sequences of the utilized PCR primersare P1: 5′ GGAAAT™ TAGCTGCAGCATCTGAC 3′ (SEQ ID NO.: 1); P2: 5′TCTAGCAAATTCGCTTGT GTTGAA 3′ (SEQ ID NO.:2); P3: 5′ CTCGCATATAGCATTAGACC3′ (SEQ ID NO.:3); and P4: 5′ CTATCTAGGCCTAAAGTAT 3′ (SEQ ID NO.:4).These regions were cloned into prokaryotic expression vectors thatexpress either fusion (pMALc and pGEX2T) or native (pET23a-c) protein tohigh levels in E. coli, and allow affinity purification of the expressedprotein on a ligand containing column.

[0308] Clostridium difficile VPI strain 10463 was obtained from the ATCC(ATCC #43255) and grown under anaerobic conditions in brain-heartinfusion medium (BBL). High molecular-weight C. difficile DNA wasisolated essentially as described by Wren and Tabaqchali (1987) J. Clin.Microbiol., 25:2402, except proteinase K and sodium dodecyl sulfate(SDS) was used to disrupt the bacteria, and cetyltrimethylammoniumbromide precipitation [as described in Ausubel et al., Current Protocolsin Molecular Biology (1989)] was used to remove carbohydrates from thecleared lysate. The integrity and yield of genomic DNA was assessed bycomparison with a serial dilution of uncut lambda DNA afterelectrophoresis on an agarose gel.

[0309] Fragments 1 and 2 were cloned by PCR, utilizing a proofreadingthermostable DNA polymerase (native pfu polymerase; Stratagene). Thehigh fidelity of this polymerase reduces the mutation problemsassociated with amplification by error prone polymerases (e.g., Taqpolymerase). PCR amplification was performed using the indicated PCRprimers (FIG. 6) in 50 μl reactions containing 10 mM Tris-HCl(8.3), 50mM KCl, 1.5 mM MgCl₂, 200 μM each dNTP, 0.2 μM each primer, and 50 ng C.difficile genomic DNA. Reactions were overlaid with 100 μl mineral oil,heated to 94° C. for 4 min, 0.5 μl native pfu polymerase (Stratagene)added, and the reaction cycled 30× at 94° C. for 1 min, 50° C. for 1min, 72° C. for 4 min, followed by 10 min at 72° C. Duplicate reactionswere pooled, chloroform extracted, and ethanol precipitated. Afterwashing in 70% ethanol, the pellets were resuspended in 50 μl TE buffer[10 mM Tris-HCL, 1 mM EDTA pH 8.0]. Aliquots of 101 μl each wererestriction digested with either EcoRI/HincII (fragment 1) or EcoRI/PstI(fragment 2), and the appropriate restriction fragments were gelpurified using the Prep-A-Gene kit (BioRad), and ligated to eitherEcoRI/SmaI-restricted pGEX2T (Pharmacia) vector (fragment 1), or theEcoRI/PstI pMAlc (New England Biolabs) vector (fragment 2). Both clonesare predicted to produce in-frame fusions with either theglutathione-S-transferase protein (pGEX vector) or the maltose bindingprotein (pMAL vector). Recombinant clones were isolated, and confirmedby restriction digestion, using standard recombinant molecular biologytechniques. [Sambrook et al., Molecular Cloning, A Laboratory Manual(1989), and designated pGA30-660 and pMA660-1100, respectively (see FIG.6 for description of the clone designations).]

[0310] Fragment 3 was cloned from a genomic library of size selectedPstI digested C. difficile genomic DNA, using standard molecular biologytechniques (Sambrook et al.). Given that the fragment 3 internal PstIsite is protected from cleavage in C. difficile genomic DNA [Price etal, Curr. Microbiol., 16:55-60 (1987)], a 4.7 kb fragment from PstIrestricted C. difficile genomic DNA was gel purified, and ligated toPstI restricted, phosphatase treated pUC9 DNA. The resulting genomiclibrary was screened with a oligonucleotide primer specific to fragment3, and multiple independent clones were isolated. The presence offragment 3 in several of these clones was confirmed by restrictiondigestion, and a clone of the indicated orientation (FIG. 6) wasrestricted with BamHI/HindIII, the released fragment purified by gelelectrophoresis, and ligated into similarly restricted pET23c expressionvector DNA (Novagen). Recombinant clones were isolated, and confirmed byrestriction digestion. This construct is predicted to create both apredicted in frame fusion with the pET protein leader sequence, as wellas a predicted C-terminal poly-histidine affinity tag, and is designatedpPA1100-2680 (see FIG. 6 for the clone designation).

[0311] b) Expression of Large Fragments of Toxin A in E. coli

[0312] Protein expression from the three expression constructs made in(a) was induced, and analyzed by Western blot analysis with an affinitypurified, goat polyclonal antiserum directed against the toxin A toxoid(Tech Lab). The procedures utilized for protein induction, SDS-PAGE, andWestern blot analysis are described in detail in Williams et al (1995),supra. In brief, 5 ml 2× YT (16 g tryptone, 10 g yeast extract, 5 g NaClper liter, pH 7.5+100 μg/ml ampicillin were added to cultures ofbacteria (BL21 for pMA1 and pGEX plasmids, and BL21(DE3)LysS for pETplasmids) containing the appropriate recombinant clone which wereinduced to express recombinant protein by addition of IPTG to 1 mM.Cultures were grown at 37° C., and induced when the cell density reached0.5 OD₆₀₀. Induced protein was allowed to accumulate for two hrs afterinduction. Protein samples were prepared by pelleting 1 ml aliquots ofbacteria by centrifugation (1 min in a microfuge), and resuspension ofthe pelleted bacteria in 150 μl of 2× SDS-PAGE sample buffer [Williamset al. (1995), supra]. The samples were heated to 95° C. for 5 min, thecooled and 5 or 10 μl aliquots loaded on 7.5% SDS-PAGE gels. BioRad highmolecular weight protein markers were also loaded, to allow estimationof the MW of identified fusion proteins. After electrophoresis, proteinwas detected either generally by staining gels with Coomassie blue, orspecifically, by blotting to nitrocellulose for Western blot detectionof specific immunoreactive protein. Western blots, (performed asdescribed in Example 3) which detect toxin A reactive protein in celllysates of induced protein from the three expression constructs areshown in FIG. 7. In this figure, lanes 1-3 contain cell lysates preparedfrom E. coli strains containing pPA1100-2860 in Bl21(DE3)lysE cells;lanes 4-6 contain cell lysates prepared from E. coli strains containingpPA1200-2860 in Bl21(DE3)lysS cells; lanes 7-9 contain cell lysatesprepared from E. coli strains containing pMA30-660; lanes 10-12 containcell lysates prepared from E. coli strains containing pMA660-1100. Thelanes were probed with an affinity purified goat antitoxin A polyclonalantibody (Tech Lab). Control lysates from uninduced cells (lanes 1, 7,and 10) contain very little immunoreactive material compared to theinduced samples in the remaining lanes. The highest molecular weightband observed for each clone is consistent with the predicted size ofthe full length fusion protein.

[0313] Each construct directs expression of high molecular weight (HMW)protein that is reactive with the toxin A antibody. The size of thelargest immunoreactive bands from each sample is consistent withpredictions of the estimated MW of the intact fusion proteins. Thisdemonstrates that the three fusions are in-frame, and that none of theclones contain cloning artifacts that disrupt the integrity of theencoded fusion protein. However, the Western blot demonstrates thatfusion protein from the two larger constructs (pGA30-660 andpPA1100-2680) are highly degraded. Also, expression levels of toxin Aproteins from these two constructs are low, since induced protein bandsare not visible by Coomassie staining (not shown). Several otherexpression constructs that fuse large sub-regions of the toxin A gene toeither pMALc or pET23a-c expression vectors, were constructed and testedfor protein induction. These constructs were made by mixing gel purifiedrestriction fragments, derived from the expression constructs shown inFIG. 6, with appropriately cleaved expression vectors, ligating, andselecting recombinant clones in which the toxin A restriction fragmentshad ligated together and into the expression vector as predicted forin-frame fusions. The expressed toxin A interval within these constructsare shown in FIG. 8, as well as the internal restriction sites utilizedto make these constructs.

[0314] As used herein, the term “interval” refers to any portion (i.e.,any segment of the toxin which is less than the whole toxin molecule) ofa clostridial toxin. In a preferred embodiment, “interval” refers toportions of C. difficile toxins such as toxin A or toxin B. It is alsocontemplated that these intervals will correspond to epitopes ofimmunologic importance, such as antigens or immunogens against which aneutralizing antibody response is effected. It is not intended that thepresent invention be limited to the particular intervals or sequencesdescribed in these Examples. It is also contemplated that sub-portionsof intervals (e.g., an epitope contained within one interval or whichbridges multiple intervals) be used as compositions and in the methodsof the present invention.

[0315] In all cases, Western blot analysis of each of these constructswith goat antitoxin A antibody (Tech Lab) detected HMW fusion protein ofthe predicted size (not shown). This confirms that the reading frame ofeach of these clones is not prematurely terminated, and is fused in thecorrect frame with the fusion partner. However, the Western blotanalysis revealed that in all cases, the induced protein is highlydegraded, and, as assessed by the absence of identifiable inducedprotein bands by Coomassie Blue staining, are expressed only at lowlevels. These results suggest that expression of high levels of intacttoxin A recombinant protein is not possible when large regions of thetoxin A gene are expressed in E. coli using these expression vectors.

[0316] c) High Level Expression of Small Toxin A Protein Fusions in E.coli

[0317] Experience indicates that expression difficulties are oftenencountered when large (greater than 100 kd) fragments are expressed inE. coli. A number of expression constructs containing smaller fragmentsof the toxin A gene were constructed, to determine if small regions ofthe gene can be expressed to high levels without extensive proteindegradation. A summary of these expression constructs are shown in FIG.9. All were constructed by in-frame fusions of convenient toxin Arestriction fragments to either the pMALc or pET23a-c vectors. Proteinpreparations from induced cultures of each of these constructs wereanalyzed by both Coomassie Blue staining and Western analysis as in (b)above. In all cases, higher levels of intact, full length fusionproteins were observed than with the larger recombinants from section(b).

[0318] d) Purification of Recombinant Toxin A Protein

[0319] Large scale (500 ml) cultures of each recombinant from (c) weregrown, induced, and soluble and insoluble protein fractions wereisolated. The soluble protein extracts were affinity chromatographed toisolate recombinant fusion protein, as described [Williams et al.(1994), supra]. In brief, extracts containing tagged pET fusions werechromatographed on a nickel chelate column, and eluted using imidazolesalts as described by the distributor (Novagen). Extracts containingsoluble pMAL fusion protein were prepared and chromatographed in columnbuffer (10 mM NaPO₄, 0.5M NaCl, 10 mM β-mercaptoethanol, pH 7.2) over anamylose resin column (New England Biolabs), and eluted with columnbuffer containing 10 mM maltose as described [Williams et al. (1995),supra]. When the expressed protein was found to be predominantlyinsoluble, insoluble protein extracts were prepared by the methoddescribed in Example 17, infra. The results are summarized in Table 16.FIG. 10 shows the sample purifications of recombinant toxin A protein.In this figure, lanes 1 and 2 contain MBP fusion protein purified byaffinity purification of soluble protein. TABLE 16 Purification OfRecombinant Toxin A Protein Yield Affinity Yield Intact Protein PurifiedSoluble % Intact Soluble Insoluble Fusion Clone^((a)) SolubilityProtein^((b)) Fusion Protein^((c)) Protein pMA30-270 Soluble   4 mg/500mls 10% NA PMA30-300 Soluble   4 mg/500 mls 5-10% NA pMA300-660Insoluble — NA  10 mg/500 ml pMA660-1100 Soluble  4.5 mg/500 mls 50% NApMA1100-1610 Soluble   18 mg/500 mls 10% NA pMA1610-1870 Both   22mg/500 mls 90%  20 mg/500 ml pMA1450-1870 Insoluble — NA 0.2 mg/500 mlpPA1100-1450 Soluble  0.1 mg/500 mls 90% NA pPA1100-1870 Soluble 0.02mg/500 mls 90% NA pMA1870-2680 Both   12 mg/500 mls 80% NA pPa1870-2680Insoluble — NA  10 mg/500 ml

[0320] Lanes 3 and 4 contain MBP fusion protein purified bysolubilization of insoluble inclusion bodies. The purified fusionprotein samples are pMA1870-2680 (lane 1), pMA660-1100 (lane 2),pMA300-600 (lane 3) and pMA1450-1870 (lane 4).

[0321] Poor yields of affinity purified protein were obtained whenpoly-histidine tagged pET vectors were used to drive expression(pPA1100-1450, pP1100-1870). However, significant protein yields wereobtained from pMAL expression constructs spanning the entire toxin Agene, and yields of full-length soluble fusion protein ranged from anestimated 200-400 μg/500 ml culture (pMA30-300) to greater than 20mg/500 ml culture (pMA1610-1870). Only one interval was expressed tohigh levels as strictly insoluble protein (pMA300-660). Thus, althoughhigh level expression was not observed when using large expressionconstructs from the toxin A gene, usable levels of recombinant proteinspanning the entire toxin A gene were obtainable by isolating inducedprotein from a series of smaller pMAL expression constructs that spanthe entire toxin A gene. This is the first demonstration of thefeasibility of expressing recombinant toxin A protein to high levels inE. coli.

[0322] e) Hemagglutination Assay Using the Toxin A Recombinant Proteins

[0323] The carboxy terminal end consisting of the repeating unitscontains the hemagglutination activity or binding domain of C. difficiletoxin A. To determine whether the expressed toxin A recombinants retainfunctional activity, hemagglutination assays were performed. Two toxin Arecombinant proteins, one containing the binding domain as eithersoluble affinity purified protein (pMA1870-2680) or SDS solubilizedinclusion body protein (pPA1870-2680) and soluble protein from oneregion outside that domain (pMA1100-1610) were tested using a describedprocedure. [H. C. Krivan et. al., Infect. Immun., 53:573 (1986).]Citrated rabbit red blood cells (RRBC)(Cocalico) were washed severaltimes with Tris-buffer (0.1M Tris and 50 mM NaCl) by centrifugation at450×g for 10 minutes at 4° C. A 1% RRBC suspension was made from thepacked cells and resuspended in Tris-buffer. Dilutions of therecombinant proteins and native toxin A (Tech Labs) were made in theTris-buffer and added in duplicate to a round-bottomed 96-wellmicrotiter plate in a final volume of 100 μl. To each well, 50 μl of the1% RRBC suspension was added, mixed by gentle tapping, and incubated at4° C. for 3-4 hours. Significant hemagglutination occurred only in therecombinant proteins containing the binding domain (pMA1870-2680) andnative toxin A. The recombinant protein outside the binding domain(pMA1100-1610) displayed no hemagglutination activity. Using equivalentprotein concentrations, the hemagglutination titer for toxin A was1:256, while titers for the soluble and insoluble recombinant proteinsof the binding domain were 1:256 and about 1:5000. Clearly, therecombinant proteins tested retained functional activity and were ableto bind RRBC's.

EXAMPLE 12 Functional Activity of IgY Reactive Against Toxin ARecombinants

[0324] The expression of recombinant toxin A protein as multiplefragments in E. coli has demonstrated the feasibility of generatingtoxin A antigen through use of recombinant methodologies (Example 11).The isolation of these recombinant proteins allows the immunoreactivityof each individual subregion of the toxin A protein to be determined(i.e., in a antibody pool directed against the native toxin A protein).This identifies the regions (if any) for which little or no antibodyresponse is elicited when the whole protein is used as a immunogen.Antibodies directed against specific fragments of the toxin A proteincan be purified by affinity chromatography against recombinant toxin Aprotein, and tested for neutralization ability. This identifies anytoxin A subregions that are essential for producing neutralizingantibodies. Comparison with the levels of immune response directedagainst these intervals when native toxin is used as an immunogenpredicts whether potentially higher titers of neutralizing antibodiescan be produced by using recombinant protein directed against aindividual region, rather than the entire protein. Finally, since it isunknown whether antibodies reactive to the recombinant toxin A proteinsproduced in Example 11 neutralize toxin A as effectively as antibodiesraised against native toxin A (Examples 9 and 10), the protectiveability of a pool of antibodies affinity purified against recombinanttoxin A fragments was assessed for its ability to neutralize toxin A.

[0325] This Example involved (a) epitope mapping of the toxin A proteinto determine the titre of specific antibodies directed againstindividual subregions of the toxin A protein when native toxin A proteinis used as an immunogen, (b) affinity purification of IgY reactiveagainst recombinant proteins spanning the toxin A gene, (c) toxin Aneutralization assays with affinity purified IgY reactive to recombinanttoxin A protein to identify subregions of the toxin A protein thatinduce the production of neutralizing antibodies, and determination ofwhether complete neutralization of toxin A can be elicited with amixture of antibodies reactive to recombinant toxin A protein.

[0326] a) Epitope Mapping of the Toxin A Gene

[0327] The affinity purification of recombinant toxin A protein specificto defined intervals of the toxin A protein allows epitope mapping ofantibody pools directed against native toxin A. This has not previouslybeen possible, since previous expression of toxin A recombinants hasbeen assessed only by Western blot analysis, without knowledge of theexpression levels of the protein [e.g., von Eichel-Streiber et al, J.Gen. Microbiol., 135:55-64 (1989)]. Thus, high or low reactivity ofrecombinant toxin A protein on Western blots may reflect proteinexpression level differences, not immunoreactivity differences. Giventhat the purified recombinant protein generated in Example 11 have beenquantitated, the issue of relative immunoreactivity of individualregions of the toxin A protein was precisely addressed.

[0328] For the purposes of this Example, the toxin A protein wassubdivided into 6 intervals (1-6), numbered from the amino (interval 1)to the carboxyl (interval 6) termini.

[0329] The recombinant proteins corresponding to these intervals werefrom expression clones (see Example 11(d) for clone designations)pMA30-300 (interval 1), pMA300-660 (interval 2), pMA660-1100 (interval3), pPA1100-1450 (interval 4), pMA1450-1870 (interval 5) andpMA1870-2680 (interval 6). These 6 clones were selected because theyspan the entire protein from amino acids numbered 30 through 2680, andsubdivide the protein into 6 small intervals. Also, the carbohydratebinding repeat interval is contained specifically in one interval(interval 6), allowing evaluation of the immune response specificallydirected against this region. Western blots of 7.5% SDS-PAGE gels,loaded and electrophoresed with defined quantities of each recombinantprotein, were probed with either goat antitoxin A polyclonal antibody(Tech Lab) or chicken antitoxin A polyclonal antibody [pCTA IgY, Example8(c)]. The blots were prepared and developed with alkaline phosphataseas previously described [Williams et al. (1995), supra]. At least 90% ofall reactivity, in either goat or chicken antibody pools, was found tobe directed against the ligand binding domain (interval 6). Theremaining immunoreactivity was directed against all five remainingintervals, and was similar in both antibody pools, except that thechicken antibody showed a much lower reactivity against interval 2 thanthe goat antibody.

[0330] This clearly demonstrates that when native toxin A is used as animmunogen in goats or chickens, the bulk of the immune response isdirected against the ligand binding domain of the protein, with theremaining response distributed throughout the remaining ⅔ of theprotein.

[0331] b) Affinity Purification of IgY Reactive Against RecombinantToxin A Protein

[0332] Affinity columns, containing recombinant toxin A protein from the6 defined intervals in (a) above, were made and used to (i) affinitypurify antibodies reactive to each individual interval from the CTA IgYpreparation [Example 8(c)], and (ii) deplete interval specificantibodies from the CTA IgY preparation. Affinity columns were made bycoupling 1 ml of PBS-washed Actigel resin (Sterogene) with regionspecific protein and {fraction (1/10)} final volume of Ald-couplingsolution (1M sodium cyanoborohydride). The total region specific proteinadded to each reaction mixture was 2.7 mg (interval 1), 3 mg (intervals2 and 3), 0.1 mg (interval 4), 0.2 mg (interval 5) and 4 mg (interval6). Protein for intervals 1, 3, and 6 was affinity purified pMA1 fusionprotein in column buffer (see Example 11). Interval 4 was affinitypurified poly-histidine containing pET fusion in PBS; intervals 2 and 5were from inclusion body preparations of insoluble pMAL fusion protein,dialyzed extensively in PBS. Aliquots of the supernatants from thecoupling reactions, before and after coupling, were assessed byCoomassie staining of 7.5% SDS-PAGE gels. Based on protein bandintensities, in all cases greater than 50% coupling efficiencies wereestimated. The resins were poured into 5 ml BioRad columns, washedextensively with PBS, and stored at 4° C.

[0333] Aliquots of the CTA IgY polyclonal antibody preparation weredepleted for each individual region as described below. A 20 ml sampleof the CTA IgY preparation [Example 8(c)] was dialyzed extensivelyagainst 3 changes of PBS (1 liter for each dialysis), quantitated byabsorbance at OD₂₈₀, and stored at 4° C. Six 1 ml aliquots of thedialyzed IgY preparation were removed, and depleted individually foreach of the six intervals. Each 1 ml aliquot was passed over theappropriate affinity column, and the eluate twice reapplied to thecolumn. The eluate was collected, and pooled with a 1 ml PBS wash. Boundantibody was eluted from the column by washing with 5 column volumes of4 M Guanidine-HCl (in 10 mM Tris-HCl, pH 8.0). The column wasreequilibrated in PBS, and the depleted antibody stock reapplied asdescribed above. The eluate was collected, pooled with a 1 ml PBS wash,quantitated by absorbance at OD₂₈₀, and stored at 4° C. In this mariner,6 aliquots of the CTA IgY preparation were individually depleted foreach of the 6 toxin A intervals, by two rounds of affinity depletion.The specificity of each depleted stock was tested by Western blotanalysis. Multiple 7.5% SDS-PAGE gels were loaded with protein samplescorresponding to all 6 toxin A subregions. After electrophoresis, thegels were blotted, and protein transfer confirmed by Ponceau S staining[protocols described in Williams et al. (1995), supra]. After blockingthe blots 1 hr at 20° C. in PBS+0.1% Tween 20 (PBST) containing 5% milk(as a blocking buffer), 4 ml of either a {fraction (1/500)} dilution ofthe dialyzed CTA IgY preparation in blocking buffer, or an equivalentamount of the six depleted antibody stocks (using OD₂₈₀ to standardizeantibody concentration) were added and the blots incubated a further 1hr at room temperature. The blots were washed and developed withalkaline phosphatase (using a rabbit anti-chicken alkaline phosphateconjugate as a secondary antibody) as previously described [Williams etal. (1995), supra]. In all cases, only the target interval was depletedfor antibody reactivity, and at least 90% of the reactivity to thetarget intervals was specifically depleted.

[0334] Region specific antibody pools were isolated by affinitychromatography as described below. Ten mls of the dialyzed CTA IgYpreparation were applied sequentially to each affinity column, such thata single 10 ml aliquot was used to isolate region specific antibodiesspecific to each of the six subregions. The columns were sequentiallywashed with 10 volumes of PBS, 6 volumes of BBS-Tween, 10 volumes ofTBS, and eluted with 4 ml Actisep elution media (Sterogene). The eluatewas dialyzed extensively against several changes of PBS, and theaffinity purified antibody collected and stored at 4° C. The volumes ofthe eluate increased to greater than 10 mls during dialysis in eachcase, due to the high viscosity of the Actisep elution media. Aliquotsof each sample were 20× concentrated using Centricon 30microconcentrators (Amicon) and stored at 4° C. The specificity of eachregion specific antibody pool was tested, relative to the dialyzed CTAIgY preparation, by Western blot analysis, exactly as described above,except that 4 ml samples of blocking buffer containing 100 μl regionspecific antibody (unconcentrated) were used instead of the depleted CTAIgY preparations. Each affinity purified antibody preparation wasspecific to the defined interval, except that samples purified againstintervals 1-5 also reacted with interval 6. This may be due tonon-specific binding to the interval 6 protein, since this proteincontains the repetitive ligand binding domain which has been shown tobind antibodies nonspecifically. [Lyerly et al., Curr. Microbiol.,19:303-306 (1989).]

[0335] The reactivity of each affinity purified antibody preparation tothe corresponding proteins was approximately the same as the reactivityof the {fraction (1/500)} diluted dialyzed CTA IgY preparation standard.Given that the specific antibody stocks were diluted {fraction (1/40)},this would indicate that the unconcentrated affinity purified antibodystocks contain {fraction (1/10)}-{fraction (1/20)} the concentration ofspecific antibodies relative to the starting CTA IgY preparation.

[0336] c) Toxin A Neutralization Assay Using Antibodies Reactive TowardRecombinant Toxin A Protein

[0337] The CHO toxin neutralization assay [Example 8(d)] was used toassess the ability of the depleted or enriched samples generated in (b)above to neutralize the cytotoxicity of toxin A. The general ability ofaffinity purified antibodies to neutralize toxin A was assessed bymixing together aliquots of all 6 concentrated stocks of the 6 affinitypurified samples generated in (b) above, and testing the ability of thismixture to neutralize a toxin A concentration of 0.1 μg/ml. The results,shown in FIG. 11, demonstrate almost complete neutralization of toxin Ausing the affinity purified (AP) mix. Some epitopes within therecombinant proteins utilized for affinity purification were probablylost when the proteins were denatured before affinity purification [byGuanidine-HCl treatment in (b) above]. Thus, the neutralization abilityof antibodies directed against recombinant protein is probablyunderestimated using these affinity purified antibody pools. Thisexperiment demonstrates that antibodies reactive to recombinant toxin Acan neutralize cytotoxicity, suggesting that neutralizing antibodies maybe generated by using recombinant toxin A protein as immunogen.

[0338] In view of the observation that the recombinant expression clonesof the toxin A gene divide the protein into 6 subregions, theneutralizing ability of antibodies directed against each individualregion was assessed. The neutralizing ability of antibodies directedagainst the ligand binding domain of toxin A was determined first.

[0339] In the toxin neutralization experiment shown in FIG. 11, interval6 specific antibodies (interval 6 contains the ligand binding domain)were depleted from the dialyzed PEG preparation, and the effect on toxinneutralization assayed. Interval 6 antibodies were depleted either byutilizing the interval 6 depleted CTA IgY preparation from (b) above(“−6 aff. depleted” in FIG. 11), or by addition of interval 6 protein tothe CTA IgY preparation (estimated to be a 10 fold molar excess overanti-interval 6 immunoglobulin present in this preparation) tocompetitively compete for interval 6 protein (“−6 prot depleted” in FIG.11). In both instances, removal of interval 6 specific antibodiesreduces the neutralization efficiency relative to the starting CTA IgYpreparation. This demonstrates that antibodies directed against interval6 contribute to toxin neutralization. Since interval 6 corresponds tothe ligand binding domain of the protein, these results demonstrate thatantibodies directed against this region in the PEG preparationcontribute to the neutralization of toxin A in this assay. However, itis significant that after removal of these antibodies, the PEGpreparation retains significant ability to neutralize toxin A (FIG. 11).This neutralization is probably due to the action of antibodies specificto other regions of the toxin A protein, since at least 90% of theligand binding region reactive antibodies were removed in the depletedsample prepared in (b) above. This conclusion was supported bycomparison of the toxin neutralization of the affinity purified (AP) mixcompared to affinity purified interval 6 antibody alone. Although someneutralization ability was observed with AP interval 6 antibodies alone,the neutralization was significantly less than that observed with themixture of all 6 AP antibody stocks (not shown).

[0340] Given that the mix of all six affinity purified samples almostcompletely neutralized the cytotoxicity of toxin A (FIG. 11), therelative importance of antibodies directed against toxin A intervals 1-5within the mixture was determined. This was assessed in two ways. First,samples containing affinity purified antibodies representing 5 of the 6intervals were prepared, such that each individual region was depletedfrom one sample. FIG. 12 demonstrates a sample neutralization curve,comparing the neutralization ability of affinity purified antibody mixeswithout interval 4 (−4) or 5 (−5) specific antibodies, relative to themix of all 6 affinity purified antibody stocks (positive control). Whilethe removal of interval 5 specific antibodies had no effect on toxinneutralization (or intervals 1-3, not shown), the loss of interval 4specific antibodies significantly reduced toxin neutralization (FIG.12).

[0341] Similar results were seen in a second experiment, in whichaffinity purified antibodies, directed against a single region, wereadded to interval 6 specific antibodies, and the effects on toxinneutralization assessed. Only interval 4 specific antibodiessignificantly enhanced neutralization when added to interval 6 specificantibodies (FIG. 13). These results demonstrate that antibodies directedagainst interval 4 (corresponding to clone pPA1100-1450 in FIG. 9) areimportant for neutralization of cytotoxicity in this assay. Epitopemapping has shown that only low levels of antibodies reactive to thisregion are generated when native toxin A is used as an immunogen[Example 12(a)]. It is hypothesized that immunization with recombinantprotein specific to this interval will elicit higher titers ofneutralizing antibodies. In summary, this analysis has identified twocritical regions of the toxin A protein against which neutralizingantibodies are produced, as assayed by the CHO neutralization assay.

EXAMPLE 13 Production and Evaluation of Avian Antitoxin Against C.difficile Recombinant Toxin A Polypeptide

[0342] In Example 12, we demonstrated neutralization of toxin A mediatedcytotoxicity by affinity purified antibodies reactive to recombinanttoxin A protein. To determine whether antibodies raised against arecombinant polypeptide fragment of C. difficile toxin A may beeffective in treating clostridial diseases, antibodies to recombinanttoxin A protein representing the binding domain were generated. Twotoxin A binding domain recombinant polypeptides, expressing the bindingdomain in either the pMALc (pMA1870-2680) or pET 23(pPA1870-2680)vector, were used as immunogens. The pMAL protein was affinity purifiedas a soluble product [Example 12(d)] and the pET protein was isolated asinsoluble inclusion bodies [Example 12(d)] and solubilized to animmunologically active protein using a proprietary method described in apending patent application (U.S. patent application Ser. No.08/129,027). This Example involves (a) immunization, (b) antitoxincollection, (c) determination of antitoxin antibody titer, (d)anti-recombinant toxin A neutralization of toxin A hemagglutinationactivity in vitro, and (e) assay of in vitro toxin A neutralizingactivity.

[0343] a) Immunization

[0344] The soluble and the inclusion body preparations each were usedseparately to immunize hens. Both purified toxin A polypeptides werediluted in PBS and emulsified with approximately equal volumes of CFAfor the initial immunization or IFA for subsequent boosterimmunizations. On day zero, for each of the recombinant preparations,two egg laying white Leghorn hens (obtained from local breeder) wereeach injected at multiple sites (intramuscular and subcutaneous) with 1ml of recombinant adjuvant mixture containing approximately 0.5 to 1.5mgs of recombinant toxin A. Booster immunizations of 1.0 mg were givenon days 14 and day 28.

[0345] b) Antitoxin Collection

[0346] Total yolk immune IgY was extracted as described in the standardPEG protocol (as in Example 1) and the final IgY pellet was dissolved insterile PBS at the original yolk volume. This material is designated“immune recombinant IgY” or “immune IgY.”

[0347] c) Antitoxin Antibody Titer

[0348] To determine if the recombinant toxin A protein was sufficientlyimmunogenic to raise antibodies in hens, the antibody titer of arecombinant toxin A polypeptide was determined by ELISA. Eggs from bothhens were collected on day 32, the yolks pooled and the antibody wasisolated using PEG as described. The immune recombinant IgY antibodytiter was determined for the soluble recombinant protein containing themaltose binding protein fusion generated in p-Mal (pMA1870-2680).Ninety-six well Falcon Pro-bind plates were coated overnight at 4° C.with 100 μL/well of toxin A recombinant at 2.5 μg/μl in PBS containing0.05% thimerosal. Another plate was also coated with maltose bindingprotein (MBP) at the same concentration, to permit comparison ofantibody reactivity to the fusion partner. The next day, the wells wereblocked with PBS containing 1% bovine serum albumin (BSA) for 1 hour at37° C. IgY isolated from immune or preimmune eggs was diluted inantibody diluent (PBS containing 1% BSA and 0.05% Tween-20), and addedto the blocked wells and incubated for 1 hour at 37° C. The plates werewashed three times with PBS with 0.05% Tween-20, then three times withPBS. Alkaline phosphatase conjugated rabbit anti-chicken IgG (Sigma)diluted 1:1000 in antibody diluent was added to the plate, and incubatedfor 1 hour at 37° C. The plates were washed as before and substrate wasadded, [p-nitrophenyl phosphate (Sigma)] at 1 mg/ml in 0.05M Na₂CO₃, pH9.5 and 10 mM MgCl₂. The plates were evaluated quantitatively on aDynatech MR 300 Micro EPA plate reader at 410 nm about 10 minutes afterthe addition of substrate.

[0349] Based on these ELISA results, high antibody titers were raised inchickens immunized with the toxin A recombinant polypeptide. Therecombinant appeared to be highly immunogenic, as it was able togenerate high antibody titers relatively quickly with few immunizations.Immune IgY titer directed specifically to the toxin A portion of therecombinant was higher than the immune IgY titer to its fusion partner,the maltose binding protein, and significantly higher than the preimmuneIgY. ELISA titers (reciprocal of the highest dilution of IgY generatinga signal) in the preimmune IgY to the MBP or the recombinant was <1:30while the immune IgY titers to MBP and the toxin A recombinant were1:18750 and >1:93750 respectively. Importantly, the anti-recombinantantibody titers generated in the hens against the recombinantpolypeptide is much higher, compared to antibodies to that region raisedusing native toxin A. The recombinant antibody titer to region 1870-2680in the CTA antibody preparation is at least five-fold lower compared tothe recombinant generated antibodies (1:18750 versus >1:93750). Thus, itappears a better immune response can be generated against a specificrecombinant using that recombinant as the immunogen compared to thenative toxin A.

[0350] This observation is significant, as it shows that becauserecombinant portions stimulate the production of antibodies, it is notnecessary to use native toxin molecules to produce antitoxinpreparations. Thus, the problems associated with the toxicity of thenative toxin are avoided and large-scale antitoxin production isfacilitated.

[0351] d) Anti-Recombinant Toxin A Neutralization of Toxin AHemagglutination Activity In Vitro

[0352] Toxin A has hemagglutinating activity besides cytotoxic andenterotoxin properties. Specifically, toxin A agglutinates rabbiterythrocytes by binding to a trisaccharide (gal 1-3B1-4GlcNAc) on thecell surface. [H. Krivan et al., Infect. Immun., 53:573-581 (1986).] Weexamined whether the anti-recombinant toxin A (immune IgY, antibodiesraised against the insoluble product expressed in pET) can neutralizethe hemagglutination activity of toxin A in vitro. The hemagglutinationassay procedure used was described by H. C. Krivan et al. Polyethyleneglycol-fractionated immune or preimmune IgY were pre-absorbed withcitrated rabbit erythrocytes prior to performing the hemagglutinationassay because we have found that IgY alone can agglutinate red bloodcells. Citrated rabbit red blood cells (RRBC's)(Cocalico) were washedtwice by centrifugation at 450×g with isotonic buffer (0.1 M Tris-HCl,0.05 M NaCl, pH 7.2). RRBC-reactive antibodies in the IgY were removedby preparing a 10% RRBC suspension (made by adding packed cells toimmune or preimmune IgY) and incubating the mixture for 1 hour at 37° C.The RRBCs were then removed by centrifugation. Neutralization of thehemagglutination activity of toxin A by antibody was tested inround-bottomed 96-well microtiter plates. Twenty-five μl of toxin A (36μg/ml) (Tech Lab) in isotonic buffer was mixed with an equal volume ofdifferent dilutions of immune or preimmune IgY in isotonic buffer, andincubated for 15 minutes at room temperature. Then, 50 μl of a 1% RRBCsuspension in isotonic buffer was added and the mixture was incubatedfor 3 hours at 4° C. Positive control wells containing the finalconcentration of 9 μg/ml of toxin A after dilution without IgY were alsoincluded. Hemagglutination activity was assessed visually, with adiffuse matrix of RRBC's coating the bottom of the well representing apositive hemagglutination reaction and a tight button of RRBC's at thebottom of the well representing a negative reaction. Theanti-recombinant immune IgY neutralized toxin A hemagglutinationactivity, giving a neutralization titer of 1:8. However, preimmune IgYwas unable to neutralize the hemagglutination ability of toxin A.

[0353] e) Assay of In Vitro Toxin A Neutralizing Activity

[0354] The ability of the anti-recombinant toxin A IgY (immune IgYantibodies raised against pMA1870-2680, the soluble recombinant bindingdomain protein expressed in pMAL, designated as Anti-tox. A-2 in FIG.14, and referred to as recombinant region 6) and pre-immune IgY,prepared as described in Example 8(c) above, to neutralize the cytotoxicactivity of toxin A was assessed in vitro using the CHO cellcytotoxicity assay, and toxin A (Tech Lab) at a concentration of 0.1μg/ml, as described in Example 8(d) above. As additional controls, theanti-native toxin A IgY (CTA) and pre-immune IgY preparations describedin Example 8(c) above were also tested. The results are shown in FIG.14.

[0355] The anti-recombinant toxin A IgY demonstrated only partialneutralization of the cytotoxic activity of toxin A, while thepre-immune IgY did not demonstrate any significant neutralizingactivity.

EXAMPLE 14 In Vivo Neutralization of C. difficile Toxin A

[0356] The ability of avian antibodies (IgY) raised against recombinanttoxin A binding domain to neutralize the enterotoxin activity of C.difficile toxin A was evaluated in vivo using Golden Syrian hamsters.The Example involved:

[0357] (a) preparation of the avian anti-recombinant toxin A IgY fororal administration;

[0358] (b) in vivo protection of hamsters from C. difficile toxin Aenterotoxicity by treatment of toxin A with avian anti-recombinant toxinA IgY; and (c) histologic evaluation of hamster ceca.

[0359] a) Preparation of the Avian Anti-Recombinant Toxin A IgY for OralAdministration

[0360] Eggs were collected from hens which had been immunized with therecombinant C. difficile toxin A fragment pMA1870-2680 (described inExample 13, above). A second group of eggs purchased at a localsupermarket was used as a pre-immune (negative) control. Egg yolkimmunoglobulin (IgY) was extracted by PEG from the two groups of eggs asdescribed in Example 8(c), and the final IgY pellets were solubilized inone-fourth the original yolk volume using 0.1M carbonate buffer (mixtureof NaHCO₃ and Na₂CO₃), pH 9.5. The basic carbonate buffer was used inorder to protect the toxin A from the acidic pH of the stomachenvironment.

[0361] b) In Vivo Protection of Hamsters Against C. difficile Toxin AEnterotoxicity by Treatment of Toxin A with Avian Anti-Recombinant ToxinA IgY

[0362] In order to assess the ability of the avian anti-recombinanttoxin A IgY, prepared in section (a) above to neutralize the in vivoenterotoxin activity of toxin A, an in vivo toxin neutralization modelwas developed using Golden Syrian hamsters. This model was based onpublished values for the minimum amount of toxin A required to elicitdiarrhea (0.08 mg toxin A/Kg body wt.) and death (0.16 mg toxin A/Kgbody wt.) in hamsters when administered orally (Lyerly et al. Infect.Immun., 47:349-352 (1985).

[0363] For the study, four separate experimental groups were used, witheach group consisting of 7 female Golden Syrian hamsters (CharlesRiver), approx. three and one-half weeks old, weighing approx. 50 gmseach. The animals were housed as groups of 3 and 4, and were offeredfood and water ad libitum through the entire length of the study.

[0364] For each animal, a mixture containing either 100 μg of toxin A(0.2 mg/Kg) or 30 μg of toxin A (0.6 mg/Kg) (C. difficile toxin A wasobtained from Tech Lab and 1 ml of either the anti-recombinant toxin AIgY or pre-immune IgY (from section (a) above) was prepared. Thesemixtures were incubated at 37° C. for 60 min. and were then administeredto the animals by the oral route. The animals were then observed for theonset of diarrhea and death for a period of 24 hrs. following theadministration of the toxin A+IgY mixtures, at the end of which time,the following results were tabulated and shown in Table 17: TABLE 17Study Outcome At 24 Hours Study Outcome at 24 Hours Experimental GroupHealthy¹ Diarrhea² Dead³ 10 μg Toxin A + 7 0 0 Antitoxin AgainstInterval 6 30 μg Toxin A + 7 0 0 Antitoxin Against Interval 6 10 μgToxin A + 0 5 2 Pre-Immune Serum 30 μg Toxin A + 0 5 2 Pre-Immune

[0365] Pretreatment of toxin A at both doses tested, using theanti-recombinant toxin A IgY, prevented all overt symptoms of disease inhamsters. Therefore, pretreatment of C. difficile toxin A, using theanti-recombinant toxin A IgY, neutralized the in vivo enterotoxinactivity of the toxin A. In contrast, all animals from the two groupswhich received toxin A which had been pretreated using pre-immune IgYdeveloped disease symptoms which ranged from diarrhea to death. Thediarrhea which developed in the animals which did not die in each of thetwo pre-immune groups, spontaneously resolved by the end of the 24 hr.study period.

[0366] c) Histologic Evaluation of Hamster Ceca

[0367] In order to further assess the ability of anti-recombinant toxinA IgY to protect hamsters from the enterotoxin activity of toxin A,histologic evaluations were performed on the ceca of hamsters from thestudy described in section (b) above.

[0368] Three groups of animals were sacrificed in order to preparehistological specimens. The first group consisted of a singlerepresentative animal taken from each of the 4 groups of survivinghamsters at the conclusion of the study described in section (b) above.These animals represented the 24 hr. timepoint of the study.

[0369] The second group consisted of two animals which were not part ofthe study described above, but were separately treated with the sametoxin A+pre-immune IgY mixtures as described for the animals in section(b) above. Both of these hamsters developed diarrhea, and weresacrificed 8 hrs. after the time of administration of the toxinA+pre-immune IgY mixtures. At the time of sacrifice, both animals werepresenting symptoms of diarrhea. These animals represented the acutephase of the study.

[0370] The final group consisted of a single untreated hamster from thesame shipment of animals as those used for the two previous groups. Thisanimal served as the normal control.

[0371] Samples of cecal tissue were removed from the 7 animals describedabove, and were fixed overnight at 4° C. using 10% buffered formalin.The fixed tissues were paraffin-embedded, sectioned, and mounted onglass microscope slides. The tissue sections were then stained usinghematoxylin and eosin (H and E stain), and were examined by lightmicroscopy.

[0372] The tissues obtained from the two 24 hr. animals which receivedmixtures containing either 10 μg or 30 μg of toxin A andanti-recombinant toxin A IgY were indistinguishable from the normalcontrol, both in terms of gross pathology, as well as at the microscopiclevel. These observations provide further evidence for the ability ofanti-recombinant toxin A IgY to effectively neutralize the in vivoenterotoxin activity of C. difficile toxin A, and thus its ability toprevent acute or lasting toxin A-induced pathology.

[0373] In contrast, the tissues from the two 24 hr. animals whichreceived the toxin A+pre-immune IgY mixtures demonstrated significantpathology. In both of these groups, the mucosal layer was observed to beless organized than in the normal control tissue. The cytoplasm of theepithelial cells had a vacuolated appearance, and gaps were presentbetween the epithelium and the underlying cell layers. The laminapropria was largely absent. Intestinal villi and crypts weresignificantly diminished, and appeared to have been overgrown by aplanar layer of epithelial cells and fibroblasts. Therefore, althoughthese animals overtly appeared to recover from the acute symptoms oftoxin A intoxication, lasting pathologic alterations to the cecal mucosahad occurred.

[0374] The tissues obtained from the two acute animals which receivedmixtures of toxin A and pre-immune IgY demonstrated the most significantpathology. At the gross pathological level, both animals were observedto have severely distended ceca which were filled with watery,diarrhea-like material. At the microscopic level, the animal that wasgiven the mixture containing 10 μg of toxin A and pre-immune IgY wasfound to have a mucosal layer which had a ragged, damaged appearance,and a disorganized, compacted quality. The crypts were largely absent,and numerous breaks in the epithelium had occurred. There was also aninflux of erythrocytes into spaces between the epithelial layer and theunderlying tissue. The animal which had received the mixture containing30 μg of toxin A and pre-immune IgY demonstrated the most severepathology. The cecal tissue of this animal had an appearance verysimilar to that observed in animals which had died from C. difficiledisease. Widespread destruction of the mucosa was noted, and theepithelial layer had sloughed. Hemorrhagic areas containing largenumbers of erythrocytes were very prevalent. All semblance of normaltissue architecture was absent from this specimen. In terms of thepresentation of pathologic events, this in vivo hamster model of toxinA−intoxication correlates very closely with the pathologic consequencesof C. difficile disease in hamsters. The results presented in thisExample demonstrate that while anti-recombinant toxin A (Interval 6) IgYis capable of only partially neutralizing the cytotoxic activity of C.difficile toxin A, the same antibody effectively neutralizes 100% of thein vivo enterotoxin activity of the toxin. While it is not intended thatthis invention be limited to this mechanism, this may be due to thecytotoxicity and enterotoxicity of C. difficile Toxin A as two separateand distinct biological functions.

EXAMPLE 15 In Vivo Neutralization of C. Difficile Toxin A by AntibodiesAgainst Recombinant Toxin A Polypeptides

[0375] The ability of avian antibodies directed against the recombinantC. difficile toxin A fragment 1870-2680 (as expressed by pMA1870-2680;see Example 13) to neutralize the enterotoxic activity of toxin A wasdemonstrated in Example 14. The ability of avian antibodies (IgYs)directed against other recombinant toxin A epitopes to neutralize nativetoxin A in vivo was next evaluated. This example involved: (a) thepreparation of IgYs against recombinant toxin A polypeptides; (b) invivo protection of hamsters against toxin A by treatment withanti-recombinant toxin A IgYs and (c) quantification of specificantibody concentration in CTA and Interval 6 IgY PEG preparations.

[0376] The nucleotide sequence of the coding region of the entire toxinA protein is listed in SEQ ID NO:5. The amino acid sequence of theentire toxin A protein is listed in SEQ ID NO:6. The amino acid sequenceconsisting of amino acid residues 1870 through 2680 of toxin A is listedin SEQ ID NO:7. The amino acid sequence consisting of amino acidresidues 1870 through 1960 of toxin A is listed in SEQ ID NO:8.

[0377] a) Preparation of IgY's Against Recombinant Toxin A Polypeptides

[0378] Eggs were collected from Leghorn hens which have been immunizedwith recombinant C. difficile toxin A polypeptide fragments encompassingthe entire toxin A protein. The polypeptide fragments used as immunogenswere: 1) pMA 1870-2680 (Interval 6), 2) pPA 1100-1450 (Interval 4), and3) a mixture of fragments consisting of pMA 30-300 (Interval 1), pMA300-660 (Interval 2), pMA 660-1100 (Interval 3) and pMA 1450-1870(Interval 5). This mixture of immunogens is referred to as Interval1235. The location of each interval within the toxin A molecule is shownin FIG. 15A. In FIG. 15A, the following abbreviations are used: pPrefers to the pET23 vector (New England BioLabs); pM refers to thepMAL™-c vector (New England BioLabs); A refers to toxin A; the numbersrefer to the amino acid interval expressed in the clone. (For example,the designation pMA30-300 indicates that the recombinant clone encodesamino acids 30-300 of toxin A and the vector used was pMAL™-c).

[0379] The recombinant proteins were generated as described in Example11. The IgYs were extracted and solubilized in 0.1M carbonate buffer pH9.5 for oral administration as described in Example 14(a). The IgYreactivities against each individual recombinant interval was evaluatedby ELISA as described in Example 13(c).

[0380] b) In Vivo Protection of Hamsters Against Toxin A by Treatmentwith Anti-Recombinant Toxin A Antibodies

[0381] The ability of antibodies raised against recombinant toxin Apolypeptides to provide in vivo protection against the enterotoxicactivity of toxin A was examined in the hamster model system. This assaywas performed as described in Example 14(b). Briefly, for each 40-50gram female Golden Syrian hamster (Charles River), 1 ml of IgY 4× (i.e.,resuspended in ¼ of the original yolk volume) PEG prep against Interval6, Interval 4 or Interval 1235 was mixed with 30 μg (LD₁₀₀ oral dose) ofC. difficile toxin A (Tech Lab). Preimmune IgY mixed with toxin A servedas a negative control. Antibodies raised against C. difficile toxoid A(Example 8) mixed with toxin A (CTA) served as a positive control. Themixture was incubated for 1 hour at 37° C. then orally administered tolightly etherized hamsters using an 18G feeding needle. The animals werethen observed for the onset of diarrhea and death for a period ofapproximately 24 hours. The results are shown in Table 18. TABLE 18Study Outcome After 24 Hours Treatment group Healthy¹ Diarrhea² Dead³Preimmune 0 0 7 CTA 5 0 0 Interval 6 6 1 0 Interval 4 0 1 6 Interval1235 0 0 7

[0382] Pre-treatment of toxin A with IgYs against Interval 6 preventeddiarrhea in 6 of 7 hamsters and completely prevented death in all 7. Incontrast, as with preimmune IgY, IgYs against Interval 4 and Interval1235 had no effect on the onset of diarrhea and death in the hamsters.

[0383] c) Quantification of Specific Antibody Concentration in CTA andInterval 6 IgY PEG Preparations

[0384] To determine the purity of IgY PEG preparations, an aliquot of apMA1870-2680 (Interval 6) IgY PEG preparation was chromatographed usingHPLC and a KW-803 sizing column (Shodex). The resulting profile ofabsorbance at 280 nm is shown in FIG. 16. The single large peakcorresponds to the predicted MW of IgY. Integration of the area underthe single large peak showed that greater than 95% of the protein elutedfrom the column was present in this single peak. This resultdemonstrated that the majority (>95%) of the material absorbing at 280nm in the PEG preparation corresponds to IgY. Therefore, absorbance at280 nm can be used to determine the total antibody concentration in PEGpreparations.

[0385] To determine the concentration of Interval 6-specific antibodies(expressed as percent of total antibody) within the CTA and pMA1870-2680(Interval 6) PEG preparations, defined quantities of these antibodypreparations were affinity purified on a pPA1870-2680(H) (shownschematically in FIG. 15B) affinity column and the specific antibodieswere quantified. In FIG. 15B the following abbreviations are used: pPrefers to the pET23 vector (New England BioLabs); pM refers to thepMAL™-c vector (New England BioLabs); pG refers to the pGEX vector(Pharmacia); pB refers to the PinPoint™ Xa vector (Promega); A refers totoxin A; the numbers refer to the amino acid interval expressed in theclone. The solid black ovals represent the MBP; the hatched ovalsrepresent glutathione S-transferase; the hatched circles represent thebiotin tag; and HHH represents the poly-histidine tag.

[0386] An affinity column containing recombinant toxin A repeat proteinwas made as follows. Four ml of PBS-washed Actigel resin (Sterogene) wascoupled with 5-10 mg of pPA1870-2680 inclusion body protein [prepared asdescribed in Example (17) and dialyzed into PBS] in a 15 ml tube(Falcon) containing {fraction (1/10)} final volume Ald-coupling solution(1 M sodium cyanoborohydride). Aliquots of the supernatant from thecoupling reactions, before and after coupling, were assessed byCoomassie staining of 7.5% SDS-PAGE gels. Based upon protein bandintensities, greater than 6 mg of recombinant protein was coupled to theresin. The resin was poured into a 10 ml column (BioRad), washedextensively with PBS, pre-eluted with 4 M guanidine-HCl (in 10 mMTris-HCl, pH 8.0; 0.005% thimerosal) and re-equilibrated with PBS. Thecolumn was stored at 4° C.

[0387] Aliquots of a pMA1870-2680 (Interval 6) or a CTA IgY polyclonalantibody preparation (PEG prep) were affinity purified on the aboveaffinity column as follows. The column was attached to an UV monitor(ISCO) and washed with PBS. For pMA1870-2680 IgY purification, a 2× PEGprep (filter sterilized using a 0.45μ filter; approximately 500 mg totalIgY) was applied. The column was washed with PBS until the baseline wasre-established (the column flow-through was saved), washed with BBSTweento elute nonspecifically binding antibodies and re-equilibrated withPBS. Bound antibody was eluted from the column in 4 M guanidine-HCl (in10 mM Tris-HCl, pH 8.0; 0.005% thimerosal). The entire elution peak wascollected in a 15 ml tube (Falcon). The column was re-equilibrated andthe column eluate was re-chromatographed as described above. Theantibody preparation was quantified by UV absorbance (the elution bufferwas used to zero the spectrophotometer). Total purified antibody wasapproximately 9 mg and 1 mg from the first and second chromatographypasses, respectively. The low yield from the second pass indicated thatmost specific antibodies were removed by the first round ofchromatography. The estimated percentage of Interval 6 specificantibodies in the pMA1870-2680 PEG prep is approximately 2%.

[0388] The percentage of Interval 6 specific antibodies in the CTA PEGprep was determined (utilizing the same column and methodology describedabove) to be approximately 0.5% of total IgY.

[0389] A 4× PEG prep contains approximately 20 mg/ml IgY. Thus in b)above, approximately 400 μg specific antibody in the Interval 6 PEG prepneutralized 30 μg toxin A in vivo.

EXAMPLE 16 In Vivo Treatment of C. difficile Disease in Hamsters byRecombinant Interval 6 Antibodies

[0390] The ability of antibodies directed against recombinant Interval 6of toxin A to protect hamsters in vivo from C. difficile disease wasexamined. This example involved: (a) prophylactic treatment of C.difficile disease and (b) therapeutic treatment of C. difficile disease.

[0391] a) Prophylactic Treatment of C. difficile Disease

[0392] This experiment was performed as described in Example 9(b). Threegroups each consisting of 7 female 100 gram Syrian hamsters (CharlesRiver) were prophylactically treated with either preimmune IgYs, IgYsagainst native toxin A and B [CTAB; see Example 8 (a) and (b)] or IgYsagainst Interval 6. IgYs were prepared as 4× PEG preparations asdescribed in Example 9(a).

[0393] The animals were orally dosed 3 times daily, roughly at 4 hourintervals, for 12 days with 1 ml antibody preparations diluted inEnsure®. Using estimates of specific antibody concentration from Example15(c), each dose of the Interval 6 antibody prep contained approximately400 μg of specific antibody. On day 2 each hamster was predisposed to C.difficile infection by the oral administration of 3.0 mg ofClindamycin-HCl (Sigma) in 1 ml of water. On day 3 the hamsters wereorally challenged with 1 ml of C. difficile inoculum strain ATCC 43596in sterile saline containing approximately 100 organisms. The animalswere then observed for the onset of diarrhea and subsequent death duringthe treatment period. The results are shown in Table 19. TABLE 19Lethality After 12 Days Of Treatment Treatment Group Number AnimalsAlive Number Animals Dead Preimmune 0 7 CTAB 6 1 Interval 6 7 0

[0394] Treatment of hamsters with orally-administered IgYs againstInterval 6 successfully protected 7 out of 7 (100%) of the animals fromC. difficile disease. One of the hamsters in this group presented withdiarrhea which subsequently resolved during the course of treatment. Asshown previously in Example 9, antibodies to native toxin A and toxin Bwere highly protective. In this Example, 6 out of 7 animals survived inthe CTAB treatment group. All of the hamsters treated with preimmunesera came down with diarrhea and died. The survivors in both the CTABand Interval 6 groups remained healthy throughout a 12 daypost-treatment period. In particular, 6 out of 7 Interval 6-treatedhamsters survived at least 2 weeks after termination of treatment whichsuggests that these antibodies provide a long-lasting cure. Theseresults represent the first demonstration that antibodies generatedagainst a recombinant region of toxin A can prevent CDAD whenadministered passively to animals. These results also indicate thatantibodies raised against Interval 6 alone may be sufficient to protectanimals from C. difficile disease when administered prophylactically.

[0395] Previously others had raised antibodies against toxin A byactively immunizing hamsters against a recombinant polypeptide locatedwithin the Interval 6 region [Lyerly, D. M., et al. (1990) Curr.Microbiol. 21:29]. FIG. 17 shows schematically the location of theLyerly, et al intra-Interval 6 recombinant protein (cloned into the pUCvector) in comparison with the complete Interval 6 construct(pMA1870-2680) used herein to generate neutralizing antibodies directedagainst toxin A. In FIG. 17, the solid black oval represents the MBPwhich is fused to the toxin A Interval 6 in pMA1870-2680.

[0396] The Lyerly, et al. antibodies (intra-Interval 6) were only ableto partially protect hamsters against C. difficile infection in terms ofsurvival (4 out of 8 animals survived) and furthermore, these antibodiesdid not prevent diarrhea in any of the animals. Additionally, animalstreated with the intra-Interval 6 antibodies [Lyerly, et al. (1990),supra] died when treatment was removed.

[0397] In contrast, the experiment shown above demonstrates that passiveadministration of anti-Interval 6 antibodies prevented diarrhea in 6 outof 7 animals and completely prevented death due to CDAD. Furthermore, asdiscussed above, passive administration of the anti-Interval 6antibodies provides a long lasting cure (i.e., treatment could bewithdrawn without incident).

[0398] b) Therapeutic Treatment of C. difficile Disease: In VivoTreatment of an Established C. difficile Infection in Hamsters withRecombinant Interval 6 Antibodies

[0399] The ability of antibodies against recombinant interval 6 of toxinA to therapeutically treat C. difficile disease was examined. Theexperiment was performed essentially as described in Example 10(b).Three groups, each containing seven to eight female Golden Syrianhamsters (100 g each; Charles River) were treated with either preimmuneIgY, IgYs against native toxin A and toxin B (CTAB) and IgYs againstInterval 6. The antibodies were prepared as described above as 4× PEGpreparations.

[0400] The hamsters were first predisposed to C. difficile infectionwith a 3 mg dose of Clindamycin-HCl (Sigma) administered orally in 1 mlof water. Approximately 24 hrs later, the animals were orally challengedwith 1 ml of C. difficile strain ATCC 43596 in sterile saline containingapproximately 200 organisms. One day after infection, the presence oftoxin A and B was determined in the feces of the hamsters using acommercial immunoassay kit (Cytoclone A+B EPA, Cambridge Biotech) toverify establishment of infection. Four members of each group wererandomly selected and tested. Feces from an uninfected hamster wastested as a negative control. All infected animals tested positive forthe presence of toxin according to the manufacturer's procedure. Theinitiation of treatment then started approximately 24 hr post-infection.

[0401] The animals were dosed daily at roughly 4 hr intervals with 1 mlantibody preparation diluted in Ensure® (Ross Labs). The amount ofspecific antibodies given per dose (determined by affinity purification)was estimated to be about 400 μg of anti-interval 6 IgY (for animals inthe Interval 6 group) and 100 μg and 70 μg of anti-toxin A (Interval6-specific) and anti-toxin B (Interval 3-specific; see Example 19),respectively, for the CTAB preparation. The animals were treated for 9days and then observed for an additional 4 days for the presence ofdiarrhea and death. The results indicating the number of survivors andthe number of dead 4 days post-infection are shown in Table 20. TABLE 20In vivo Therapeutic Treatment With Interval 6 Antibodies Treatment GroupNumber Animals Alive Number Animals Dead Preimmune 4 3 CTAB 8 0 Interval6 8 0

[0402] Antibodies directed against both Interval 6 and CTAB successfullyprevented death from C. difficile when therapeutically administered 24hr after infection. This result is significant since many investigatorsbegin therapeutic treatment of hamsters with existing drugs (e.g.,vancomycin, phenelfamycins, tiacumicins, etc.) 8 hr post-infection[Swanson, et al. (1991) Antimicrobial Agents and Chemotherapy 35:1108and (1989) J. Antibiotics 42:94].

[0403] Forty-two percent of hamsters treated with preimmune IgY diedfrom CDAD. While the anti-Interval 6 antibodies prevented death in thetreated hamsters, they did not eliminate all symptoms of CDAD as 3animals presented with slight diarrhea. In addition, one CTAB-treatedand one preimmune-treated animal also had diarrhea 14 dayspost-infection. These results indicate that anti-Interval 6 antibodiesprovide an effective means of therapy for CDAD.

EXAMPLE 17 Induction of Toxin A Neutralizing Antibodies Requires SolubleInterval 6 Protein

[0404] As shown in Examples 11(d) and 15, expression of recombinantproteins in E. coli may result in the production of either soluble orinsoluble protein. If insoluble protein is produced, the recombinantprotein is solubilized prior to immunization of animals. To determinewhether, one or both of the soluble or insoluble recombinant proteinscould be used to generate neutralizing antibodies to toxin A, thefollowing experiment was performed. This example involved a) expressionof the toxin A repeats and subfragments of these repeats in E. coliusing a variety of expression vectors; b) identification of recombinanttoxin A repeats and sub-regions to which neutralizing antibodies bind;and c) determination of the neutralization ability of antibodies raisedagainst soluble and insoluble toxin A repeat immunogen.

[0405] a) Expression of the Toxin A Repeats and Subfragments of theseRepeats in E. coli Using a Variety of Expression Vectors

[0406] The Interval 6 immunogen utilized in Examples 15 and 16 was thepMA1870-2680 protein, in which the toxin A repeats are expressed as asoluble fusion protein with the MBP (described in Example 11).Interestingly, expression of this region (from the SpeI site to the endof the repeats, see FIG. 15B) in three other expression constructs, aseither native (pPA1870-2680), poly-His tagged [pPA1870-2680 (H)] orbiotin-tagged (pBA1870-2680) proteins resulted in completely insolubleprotein upon induction of the bacterial host (see FIG. 15B). The hoststrain BL21 (Novagen) was used for expression of pBA1870-2680 and hoststrain BL21(DE3) (Novagen) was used for expression of pPA1870-2680 andpPA1870-2680(H). These insoluble proteins accumulated to high levels ininclusion bodies. Expression of recombinant plasmids in E. coli hostcells grown in 2× YT medium was performed as described [Williams, et al(1995), supra].

[0407] As summarized in FIG. 15B, expression of fragments of the toxin Arepeats (as either N-terminal SpeI-EcoRI fragments, or C-terminalEcoRI-end fragments) also yielded high levels of insoluble protein usingpGEX (pGA1870-2190), PinPoint™-Xa (pBA1870-2190 and pBA2250-2680) andpET expression systems (pPA1870-2190). The pGEX and pET expressionsystems are described in Example 11. The PinPoint™-Xa expression systemdrives the expression of fusion proteins in E. coli. Fusion proteinsfrom PinPoint™-Xa vectors contain a biotin tag at the amino-terminal endand can be affinity purified SoftLink™ Soft Release avidin resin(Promega) under mild denaturing conditions (5 mM biotin).

[0408] The solubility of expressed proteins from the pPG1870-2190 andpPA1870-2190 expression constructs was determined after induction ofrecombinant protein expression under conditions reported to enhanceprotein solubility [These conditions comprise growth of the host atreduced temperature (30° C.) and the utilization of high (1 mM IPTG) orlow (0.1 mM IPTG) concentrations of inducer [Williams et al. (1995),supra]. All expressed recombinant toxin A protein was insoluble underthese conditions. Thus, expression of these fragments of the toxin Arepeats in pET and pGEX expression vectors results in the production ofinsoluble recombinant protein even when the host cells are grown atreduced temperature and using lower concentrations of the inducer.Although expression of these fragments in pMa1 vectors yielded affinitypurifiable soluble fusion protein, the protein was either predominantlyinsoluble (pMA1870-2190) or unstable (pMA2250-2650). Attempts tosolubilize expressed protein from the pMA1870-2190 expression constructusing reduced temperature or lower inducer concentration (as describedabove) did not improve fusion protein solubility.

[0409] Collectively, these results demonstrate that expression of thetoxin A repeat region in E. coli results in the production of insolublerecombinant protein, when expressed as either large (aa 1870-2680) orsmall (aa 1870-2190 or aa 2250-2680) fragments, in a variety ofexpression vectors (native or poly-his tagged pET, pGEX or PinPoint™-Xavectors), utilizing growth conditions shown to enhance proteinsolubility. The exception to this rule were fusions with the MBP, whichenhanced protein solubility, either partially (pMA1870-2190) or fully(pMA1870-2680).

[0410] b) Identification Of Recombinant Toxin A Repeats and Sub-Regionsto which Neutralizing Antibodies Bind

[0411] Toxin A repeat regions to which neutralizing antibodies bind wereidentified by utilizing recombinant toxin A repeat region proteinsexpressed as soluble or insoluble proteins to deplete protectiveantibodies from a polyclonal pool of antibodies against native C.difficile toxin A. An in vivo assay was developed to evaluate proteinsfor the ability to bind neutralizing antibodies.

[0412] The rational for this assay is as follows. Recombinant proteinswere first pre-mixed with antibodies against native toxin A (CTAantibody; generated in Example 8) and allowed to react. Subsequently, C.difficile toxin A was added at a concentration lethal to hamsters andthe mixture was administered to hamsters via IP injection. If therecombinant protein contains neutralizing epitopes, the CTA antibodieswould lose their ability to bind toxin A resulting in diarrhea and/ordeath of the hamsters.

[0413] The assay was performed as follows. The lethal dose of toxin Awhen delivered orally to nine 40 to 50 g Golden Syrian hamsters (Sasco)was determined to be 10 to 30 μg. The PEG-purified CTA antibodypreparation was diluted to 0.5× concentration (i.e., the antibodies werediluted at twice the original yolk volume) in 0.1 M carbonate buffer, pH9.5. The antibodies were diluted in carbonate buffer to protect themfrom acid degradation in the stomach. The concentration of 0.5× was usedbecause it was found to be the lowest effective concentration againsttoxin A. The concentration of Interval 6-specific antibodies in the 0.5×CTA prep was estimated to be 10-15 μg/ml (estimated using the methoddescribed in Example 15).

[0414] The inclusion body preparation [insoluble Interval 6 protein;pPA1870-2680(H)] and the soluble Interval 6 protein [pMA1870-2680; seeFIG. 15] were both compared for their ability to bind to neutralizingantibodies against C. difficile toxin A (CTA). Specifically, 1 to 2 mgof recombinant protein was mixed with 5 ml of a 0.5× CTA antibody prep(estimated to contain 60-70 μg of Interval 6-specific antibody). Afterincubation for 1 hr at 37° C., CTA (Tech Lab) at a final concentrationof 30 μg/ml was added and incubated for another 1 hr at 37° C. One ml ofthis mixture containing 30 μg of toxin A (and 10-15 μg of Interval6-specific antibody) was administered orally to 40-50 g Golden Syrianhamsters (Sasco). Recombinant proteins that result in the loss ofneutralizing capacity of the CTA antibody would indicate that thoseproteins contain neutralizing epitopes. Preimmune and CTA antibodies(both at 0.5×) without the addition of any recombinant protein served asnegative and positive controls, respectively.

[0415] Two other inclusion body preparations, both expressed asinsoluble products in the pET vector, were tested; one containing adifferent insert (toxin B fragment) other than Interval 6 calledpPB1850-2070 (see FIG. 18) which serves as a control for insolubleInterval 6, the other was a truncated version of the Interval 6 regioncalled pPA1870-2190 (see FIG. 15B). The results of this experiment areshown in Table 21. TABLE 21 Binding Of Neutralizing Antibodies BySoluble Interval 6 Protein Study Outcome After 24 Hours Treatment Group¹Healthy² Diarrhea³ Dead⁴ Preimmune Ab 0 3 2 CTA Ab 4 1 0 CTA Ab + Int 6(soluble) 1 2 2 CTA Ab + Int 6 (insoluble) 5 0 0 CTA Ab + pPB1850-2070 50 0 CTA Ab + pPA1870-2190 5 0 0

[0416] Preimmune antibody was ineffective against toxin A, whileanti-CTA antibodies at a dilute 0.5× concentration almost completelyprotected the hamsters against the enterotoxic effects of CTA. Theaddition of recombinant proteins pPB1850-2070 or pPA1870-2190 to theanti-CTA antibody had no effect upon its protective ability, indicatingthat these recombinant proteins do not bind to neutralizing antibodies.On the other hand, recombinant Interval 6 protein was able to bind toneutralizing anti-CTA antibodies and neutralized the in vivo protectiveeffect of the anti-CTA antibodies. Four out of five animals in the grouptreated with anti-CTA antibodies mixed with soluble Interval 6 proteinexhibited toxin associated toxicity (diarrhea and death). Moreover, theresults showed that Interval 6 protein must be expressed as a solubleproduct in order for it to bind to neutralizing anti-CTA antibodiessince the addition of insoluble Interval 6 protein had no effect on theneutralizing capacity of the CTA antibody prep.

[0417] c) Determination of Neutralization Ability of Antibodies RaisedAgainst Soluble and Insoluble Toxin A Repeat Immunogen

[0418] To determine if neutralizing antibodies are induced againstsolubilized inclusion bodies, insoluble toxin A repeat protein wassolubilized and specific antibodies were raised in chickens. InsolublepPA1870-2680 protein was solubilized using the method described inWilliams et al. (1995), supra. Briefly, induced cultures (500 ml) werepelleted by centrifugation at 3,000×g for 10 min at 4° C. The cellpellets were resuspended thoroughly in 10 ml of inclusion bodysonication buffer (25 mM HEPES pH 7.7, 100 mM KCl, 12.5 mM MgCl₂, 20%glycerol, 0.1% (v/v) Nonidet P-40, 1 mM DTT). The suspension wastransferred to a 30 ml non-glass centrifuge tube. Five hundred μl of 10mg/ml lysozyme was added and the tubes were incubated on ice for 30 min.The suspension was then frozen at −70° C. for at least 1 hr. Thesuspension was thawed rapidly in a water bath at room temperature andthen placed on ice. The suspension was then sonicated using at leasteight 15 sec bursts of the microprobe (Branson Sonicator Model No. 450)with intermittent cooling on ice.

[0419] The sonicated suspension was transferred to a 35 ml Oakridge tubeand centrifuged at 6,000×g for 10 min at 4° C. to pellet the inclusionbodies. The pellet was washed 2 times by pipetting or vortexing infresh, ice-cold RIPA buffer [0.1% SDS, 1% Triton X-100, 1% sodiumdeoxycholate in TBS (25 mM Tris-Cl pH 7.5, 150 mM NaCl)]. The inclusionbodies were recentrifuged after each wash. The inclusion bodies weredried and transferred using a small metal spatula to a 15 ml tube(Falcon). One ml of 10% SDS was added and the pellet was solubilized bygently pipetting the solution up and down using a 1 ml micropipettor.The solubilization was facilitated by heating the sample to 95° C. whennecessary.

[0420] Once the inclusion bodies were in solution, the samples werediluted with 9 volumes of PBS. The protein solutions were dialyzedovernight against a 100-fold volume of PBS containing 0.05% SDS at roomtemperature. The dialysis buffer was then changed to PBS containing0.01% SDS and the samples were dialyzed for several hours to overnightat room temperature. The samples were stored at 4° C. until used. Priorto further use, the samples were warmed to room temperature to allow anyprecipitated SDS to go back into solution.

[0421] The inclusion body preparation was used to immunize hens. Theprotein was dialyzed into PBS and emulsified with approximately equalvolumes of CFA for the initial immunization or IFA for subsequentbooster immunizations. On day zero, for each of the recombinantrecombinant preparations, two egg laying white Leghorn hens were eachinjected at multiple sites (IM and SC) with 1 ml of recombinantprotein-adjuvant mixture containing approximately 0.5-1.5 mg ofrecombinant protein. Booster immunizations of 1.0 mg were given of days14 and day 28. Eggs were collected on day 32 and the antibody isolatedusing PEG as described in Example 14(a). High titers of toxin A specificantibodies were present (as assayed by ELISA, using the method describedin Example 13). Titers were determined for both antibodies againstrecombinant polypeptides pPA1870-2680 and pMA1870-2680 and were found tobe comparable at >1:62,500.

[0422] Antibodies against soluble Interval 6 (pMA1870-2680) andinsoluble Interval 6 [(inclusion body), pPA1870-2680] were tested forneutralizing ability against toxin A using the in vivo assay describedin Example 15(b). Preimmune antibodies and antibodies against toxin A(CTA) served as negative and positive controls, respectively. Theresults are shown in Table 22. TABLE 22 Neutralization Of Toxin A ByAntibodies Against Soluble Interval 6 Protein Study Outcome After 24Hours Antibody Treatment Group Healthy¹ Diarrhea² Dead³ Preimmune 1 0 4CTA 5 0 0 Interval 6 (Soluble)⁴ 5 0 0 Interval 6 (Insoluble) 0 2 3

[0423] Antibodies raised against native toxin A were protective whilepreimmune antibodies had little effect. As found using the in vitro CHOassay [described in Example 8(d)] where antibodies raised against thesoluble Interval 6 could partially neutralize the effects of toxin A,here they were able to completely neutralize toxin A in vivo. Incontrast, the antibodies raised against the insoluble Interval 6 wasunable to neutralize the effects of toxin A in vivo as shown above(Table 22) and in vitro as shown in the CHO assay [described in Example8(d)].

[0424] These results demonstrate that soluble toxin A repeat immunogenis necessary to induce the production of neutralizing antibodies inchickens, and that the generation of such soluble immunogen is obtainedonly with a specific expression vector (pMa1) containing the toxin Aregion spanning aa 1870-2680. That is to say, insoluble protein that issubsequently solubilized does not result in a toxin A antigen that willelicit a neutralizing antibody.

EXAMPLE 18 Cloning and Expression of the C. difficile Toxin B Gene

[0425] The toxin B gene has been cloned and sequenced; the amino acidsequence deduced from the cloned nucleotide sequence predicts a MW of269.7 kD for toxin B [Barroso et al., Nucl. Acids Res. 18:4004 (1990)].The nucleotide sequence of the coding region of the entire toxin B geneis listed in SEQ ID NO:9. The amino acid sequence of the entire toxin Bprotein is listed in SEQ ID NO:10 The amino acid sequence consisting ofamino acid residues 1850 through 2360 of toxin B is listed in SEQ IDNO:11. The amino acid sequence consisting of amino acid residues 1750through 2360 of toxin B is listed in SEQ ID NO:12.

[0426] Given the expense and difficulty of isolating native toxin Bprotein, it would be advantageous to use simple and inexpensiveprocaryotic expression systems to produce and purify high levels ofrecombinant toxin B protein for immunization purposes. Ideally, theisolated recombinant protein would be soluble in order to preservenative antigenicity, since solubilized inclusion body proteins often donot fold into native conformations. Indeed as shown in Example 17,neutralizing antibodies against recombinant toxin A were only obtainedwhen soluble recombinant toxin A polypeptides were used as theimmunogen. To allow ease of purification, the recombinant protein shouldbe expressed to levels greater than 1 mg/liter of E. coli culture.

[0427] To determine whether high levels of recombinant toxin B proteincould be produced in E. coli, fragments of the toxin B gene were clonedinto various prokaryotic expression vectors, and assessed for theability to express recombinant toxin B protein in E. coli. This Exampleinvolved (a) cloning of the toxin B gene and (b) expression of the toxinB gene in E. coli.

[0428] a) Cloning of the Toxin B Gene

[0429] The toxin B gene was cloned using PCR amplification from C.difficile genomic DNA. Initially, the gene was cloned in two overlappingfragments, using primer pairs P5/P6 and P7/P8. The location of theseprimers along the toxin B gene is shown schematically in FIG. 18. Thesequence of each of these primers is: P5: 5′ TAGAAAAAATGGCAAATGT 3′;(SEQ ID NO:11) P6: 5′ TTTCATCTTGTAGAGTCAAAG 3′; (SEQ ID NO:12) P7:5′ GATGCCACAAGATGATTTAGTG 3′; (SEQ ID NO:13) and P8:5′ CTAATTGAGCTGTATCAGGATC 3′. (SEQ ID NO:14)

[0430]FIG. 18 also shows the location of the following primers along thetoxin B gene: P9 which consists of the sequence 5′CGGAATTCCTAGAAAAAATGGCAA ATG 3′ (SEQ ID NO:15); P10 which consists ofthe sequence 5′ GCTCTAGAATGA CCATAAGCTAGCCA 3′ (SEQ ID NO:16); P11 whichconsists of the sequence 5′ CGGAATTCGAGTTGGTAGAAAGGTGGA 3′ (SEQ ID NO:17); P13 which consists of the sequence 5′ CGGAATTCGGTTATTATCTTAAGGATG3′ (SEQ ID NO:18); and P14 which consists of the sequence 5′CGGAATTCTTGATAACTGGAT TTGTGAC 3′ (SEQ ID NO:19). The amino acid sequenceconsisting of amino acid residues 1852 through 2362 of toxin B is listedin SEQ ID NO:20. The amino acid sequence consisting of amino acidresidues 1755 through 2362 of toxin B is listed in SEQ ID NO:21.

[0431]Clostridium difficile VPI strain 10463 was obtained from theAmerican Type Culture Collection (ATCC 43255) and grown under anaerobicconditions in brain-heart infusion medium (Becton Dickinson). Highmolecular-weight C. difficile DNA was isolated essentially as described[Wren and Tabaqchali (1987) J. Clin. Microbiol., 25:2402], except 1) 100μg/ml proteinase K in 0.5% SDS was used to disrupt the bacteria and 2)cetytrimethylammoniun bromide (CTAB) precipitation [as described byAusubel et al, Eds., Current Protocols in Molecular Biology, Vol. 2(1989) Current Protocols] was used to remove carbohydrates from thecleared lysate. Briefly, after disruption of the bacteria withproteinase K and SDS, the solution is adjusted to approximately 0.7 MNaCl by the addition of a {fraction (1/7)} volume of 5M NaCl. A{fraction (1/10)} volume of CTAB/NaCl (10% CTAB in 0.7 M NaCl) solutionwas added and the solution was mixed thoroughly and incubated 10 min at65° C. An equal volume of chloroform/isoamyl alcohol (24:1) was addedand the phases were thoroughly mixed. The organic and aqueous phaseswere separated by centrifugation in a microfuge for 5 min. The aqueoussupernatant was removed and extracted with phenol/chloroform/isoamylalcohol (25:24:1). The phases were separated by centrifugation in amicrofuge for 5 min. The supernatant was transferred to a fresh tube andthe DNA was precipitated with isopropanol. The DNA precipitate waspelleted by brief centrifugation in a microfuge. The DNA pellet waswashed with 70% ethanol to remove residual CTAB. The DNA pellet was thendried and redissolved in TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA).The integrity and yield of genomic DNA was assessed by comparison with aserial dilution of uncut lambda DNA after electrophoresis on an agarosegel.

[0432] Toxin B fragments were cloned by PCR utilizing a proofreadingthermostable DNA polymerase [native Pfu polymerase (Stratagene)]. Thehigh fidelity of this polymerase reduces the mutation problemsassociated with amplification by error prone polymerases (e.g., Taqpolymerase). PCR amplification was performed using the PCR primer pairsP5 (SEQ ID NO:11) with P6 (SEQ ID NO:12) and P7 (SEQ ID NO: 13) with P8(SEQ ID NO:14) in 50 μl reactions containing 10 mM Tris-HCl pH 8.3, 50mM KCl, 1.5 mM MgCl₂, 200 μM of each dNTP, 0.2 μM each primer, and 50 ngC. difficile genomic DNA. Reactions were overlaid with 100 μl mineraloil, heated to 94° C. for 4 min, 0.5 μl native Pfu polymerase(Stratagene) was added, and the reactions were cycled 30 times at 94° C.for 1 min, 50° C. for 1 min, 72° C. (2 min for each kb of sequence to beamplified), followed by 10 min at 72° C. Duplicate reactions werepooled, chloroform extracted, and ethanol precipitated. After washing in70% ethanol, the pellets were resuspended in 50 μl TE buffer (10 mMTris-HCl pH 8.0, 1 mM EDTA).

[0433] The P5/P6 amplification product was cloned into pUC19 as outlinedbelow. 10 μl aliquots of DNA were gel purified using the Prep-a-Gene kit(BioRad), and ligated to SmaI restricted pUC19 vector. Recombinantclones were isolated and confirmed by restriction digestion usingstandard recombinant molecular biology techniques (Sambrook et al.,1989). Inserts from two independent isolates were identified in whichthe toxin B insert was oriented such that the vector BamHI and SacIsites were 5′ and 3′ oriented, respectively (pUCB10-1530). Theinsert-containing BamHI/SacI fragment was cloned into similarly cutpET23a-c vector DNA, and protein expression was induced in small scalecultures (5 ml) of identified clones.

[0434] Total protein extracts were isolated, resolved on SDS-PAGE gels,and toxin B protein identified by Western analysis utilizing a goatanti-toxin B affinity purified antibody (Tech Lab). Procedures forprotein induction, SDS-PAGE, and Western blot analysis were performed asdescribed in Williams et al. (1995), supra. In brief, 5 ml cultures ofbacteria grown in 2XYT containing 100 μg/ml ampicillin containing theappropriate recombinant clone were induced to express recombinantprotein by addition of IPTG to 1 mM. The E. coli hosts used were:BL21(DE3) or BL21(DE3)LysS (Novagen) for pET plasmids.

[0435] Cultures were induced by the addition of IPTG to a finalconcentration of 1.0 mM when the cell density reached 0.5 OD₆₀₀, andinduced protein was allowed to accumulate for two hrs after induction.Protein samples were prepared by pelleting 1 ml aliquots of bacteria bycentrifugation (1 min in microfuge), and resuspension of the pelletedbacteria in 150 μl of 2× SDS-PAGE sample buffer (0.125 mM Tris-HCl pH6.8, 2 mM EDTA, 6% SDS, 20% glycerol, 0.025% bromophenol blue;β-mercaptoethanol is added to 5% before use). The samples were heated to95° C. for 5 min, then cooled and 5 or 10 μls loaded on 7.5% SDS-PAGEgels. High molecular weight protein markers (BioRad) were also loaded,to allow estimation of the MW of identified fusion proteins. Afterelectrophoresis, protein was detected either generally by staining thegels with Coomassie Blue, or specifically, by blotting to nitrocellulosefor Western blot detection of specific immunoreactive protein. The MW ofinduced toxin B reactive protein allowed the integrity of the toxin Breading frame to be determined.

[0436] The pET23b recombinant (pPB10-1530) expressed high MW recombinanttoxin B reactive protein, consistent with predicted values. Thisconfirmed that frame terminating errors had not occurred during PCRamplification. A pET23b expression clone containing the 10-1750aainterval of the toxin B gene was constructed, by fusion of theEcoRV-SpeI fragment of the P7/P8 amplification product to the P5-EcoRVinterval of the P5/P6 amplification product (see FIG. 18) in pPB10-1530.The integrity of this clone (pPB10-1750) was confirmed by restrictionmapping, and Western blot detection of expressed recombinant toxin Bprotein. Levels of induced protein from both pPB10-1530 and pPB10-1750were too low to facilitate purification of usable amounts of recombinanttoxin B protein. The remaining 1750-2360 aa interval was directly clonedinto pMAL or pET expression vectors from the P7/P8 amplification productas described below.

[0437] b) Expression of the Toxin B Gene

[0438] i) Overview of Expression Methodologies

[0439] Fragments of the toxin B gene were expressed as either native orfusion proteins in E. coli. Native proteins were expressed in either thepET23a-c or pET16b expression vectors (Novagen). The pET23 vectorscontain an extensive polylinker sequence in all three reading frames(a-c vectors) followed by a C-terminal poly-histidine repeat. The pET16bvector contains a N-terminal poly-histidine sequence immediately 5′ to asmall polylinker. The poly-histidine sequence binds to Ni-Chelatecolumns and allows affinity purification of tagged target proteins[Williams et al. (1995), supra]. These affinity tags are small (10 aafor pET16b, 6 aa for pET23) allowing the expression and affinitypurification of native proteins with only limited amounts of foreignsequences.

[0440] An N-terminal histidine-tagged derivative of pET16b containing anextensive cloning cassette was constructed to facilitate cloning ofN-terminal poly-histidine tagged toxin B expressing constructs. This wasaccomplished by replacement of the promoter region of the pET23a and bvectors with that of the pET16b expression vector. Each vector wasrestricted with BglII and NdeI, and the reactions resolved on a 1.2%agarose gel. The pET16b promoter region (contained in a 200 bpBglII-NdeI fragment) and the promoter-less pET23 a or b vectors were cutfrom the gel, mixed and Prep-A-Gene (BioRad) purified. The eluted DNAwas ligated, and transformants screened for promoter replacement by NcoIdigestion of purified plasmid DNA (the pET16b promoter contains thissite, the pET23 promoter does not). These clones (denoted pETHisa or b)contain the pET16b promoter (consisting of a pT7-lac promoter, ribosomebinding site and poly-histidine (10aa) sequence) fused at the NdeI siteto the extensive pET23 polylinker.

[0441] All MBP fusion proteins were constructed and expressed in thepMAL™-c or pMAL™-p2 vectors (New England Biolabs) in which the proteinof interest is expressed as a C-terminal fusion with MBP. All pETplasmids were expressed in either the BL21(DE3) or BL21(DE3)LysSexpression hosts, while pMa1 plasmids were expressed in the BL21 host.

[0442] Large scale (500 mls to 1 liter) cultures of each recombinantwere grown in 2×YT broth, induced, and soluble protein fractions wereisolated as described [Williams, et al. (1995), supra]. The solubleprotein extracts were affinity chromatographed to isolate recombinantfusion protein, as described [Williams et al., (1995) supra]. In brief,extracts containing tagged pET fusions were chromatographed on a nickelchelate column, and eluted using imidazole salts or low pH (pH 4.0) asdescribed by the distributor (Novagen or Qiagen). Extracts containingsoluble pMAL fusion protein were prepared and chromatographed in PBSbuffer over an amylose resin (New England Biolabs) column, and elutedwith PBS containing 10 mM maltose as described [Williams et al. (1995),supra].

[0443] ii) Overview of Toxin B Expression

[0444] In both large expression constructs described in (a) above, onlylow level (i.e., less than 1 mg/liter of intact or nondegradedrecombinant protein) expression of recombinant protein was detected. Anumber of expression constructs containing smaller fragments of thetoxin B gene were then constructed, to determine if small regions of thegene can be expressed to high levels (i.e., greater than 1 mg/literintact protein) without extensive protein degradation. All wereconstructed by in frame fusions of convenient toxin B restrictionfragments to either the pMAL-c, pET23a-c, pET16b or pETHisa-b expressionvectors, or by engineering restriction sites at specific locations usingPCR amplification [using the same conditions described in (a) above]. Inall cases, clones were verified by restriction mapping, and, whereindicated, DNA sequencing.

[0445] Protein preparations from induced cultures of each of theseconstructs were analyzed, by SDS-PAGE, to estimate protein stability(Coomassie Blue stairung) and immunoreactivity against anti-toxin Bspecific antiserum (Western analysis). Higher levels of intact (i.e.,nondegraded), full length fusion proteins were observed with the smallerconstructs as compared with the larger recombinants, and a series ofexpression constructs spanning the entire toxin B gene were constructed(FIGS. 18, 19 and 20 and Table 23).

[0446] Constructs that expressed significant levels of recombinant toxinB protein (greater than 1 mg/liter intact recombinant protein) in E coliwere identified and a series of these clones that spans the toxin B geneare shown in FIG. 19 and summarized in Table 23. These clones wereutilized to isolate pure toxin B recombinant protein from the entiretoxin B gene. Significant protein yields were obtained from pMALexpression constructs spanning the entire toxin B gene, and yields offull length soluble fusion protein ranged from an estimated 1 mg/literculture (pMB1100-1530) to greater than 20 mg/liter culture(pMB1750-2360).

[0447] Representative purifications of MBP and poly-histidine-taggedtoxin B recombinants are shown in FIGS. 21 and 22. FIG. 21 shows aCoomassie Blue stained 7.5% SDS-PAGE gel on which various proteinsamples extracted from bacteria harboring pMB1850-2360 wereelectrophoresed. Samples were loaded as follows: Lane 1: proteinextracted from uninduced culture; Lane 2: induced culture protein; Lane3: total protein from induced culture after sonication; Lane 4: solubleprotein; and Lane 5: eluted affinity purified protein. FIG. 22 depictsthe purification of recombinant proteins expressed in bacteria harboringeither pPB1850-2360 (Lanes 1-3) or pPB1750-2360 (Lanes 4-6). Sampleswere loaded as follows: uninduced total protein (Lanes 1 and 4); inducedtotal protein (Lanes 2 and 5); and eluted affinity purified protein(Lanes 3 and 6). The broad range molecular weight protein markers(BioRad) are shown in Lane 7.

[0448] Thus, although high level expression was not attained using largeexpression constructs from the toxin B gene, usable levels ofrecombinant protein were obtained by isolating induced protein from aseries of smaller pMAL expression constructs that span the entire toxinB gene.

[0449] These results represent the first demonstration of thefeasibility of expressing recombinant toxin B protein to high levels inE. coli. As well, expression of small regions of the putative ligandbinding domain (repeat region) of toxin B as native protein yieldedinsoluble protein, while large constructs, or fusions to MBP weresoluble (FIG. 19), demonstrating that specific methodologies arenecessary to produce soluble fusion protein from this interval.

[0450] iii) Clone Construction and Expression Details

[0451] A portion of the toxin B gene containing the toxin B repeatregion [amino acid residues 1852-2362 of toxin B (SEQ ID NO:20)] wasisolated by PCR amplification of this interval of the toxin B gene fromC. difficile genomic DNA. The sequence, and location within the toxin Bgene, of the two PCR primers [P7 (SEQ ID NO:13) and P8 (SEQ ID NO: 14)]used to amplify this region are shown in FIG. 18.

[0452] DNA from the PCR amplification was purified by chloroformextraction and ethanol precipitation as described above. The DNA wasrestricted with SpeI, and the cleaved DNA was resolved by agarose gelelectrophoresis. The restriction digestion with SpeI cleaved the 3.6 kbamplification product into a 1.8 kb doublet band. This doublet band wascut from the gel and mixed with appropriately cut, gel purified pMALc orpET23b vector. These vectors were prepared by digestion with HindIII,filling in the overhanging ends using the Klenow enzyme, and cleavingwith XbaI (pMALc) or NheI (pET23b). The gel purified DNA fragments werepurified using the Prep-A-Gene kit (BioRad) and the DNA was ligated,transformed and putative recombinant clones analyzed by restrictionmapping.

[0453] pET and pMa1 clones containing the toxin B repeat insert (aainterval 1750-2360 of toxin B) were verified by restriction mapping,using enzymes that cleaved specific sites within the toxin B region. Inboth cases fusion of the toxin B SpeI site with either the compatibleXbaI site (pMa1) or compatible NheI site (pET) is predicted to create anin frame fusion. This was confirmed in the case of the pMB1750-2360clone by DNA sequencing of the clone junction and 5′ end of the toxin Binsert using a MBP specific primer (New England Biolabs). In the case ofthe pET construct, the fusion of the blunt ended toxin B 3′ end to thefilled HindIII site should create an in-frame fusion with the C-terminalpoly-histidine sequence in this vector. The pPB1750-2360 clone selectedhad lost, as predicted, the HindIII site at this clone junction; thiseliminated the possibility that an additional adenosine residue wasadded to the 3′ end of the PCR product by a terminal transferaseactivity of the Pfu polymerase, since fusion of this adenosine residueto the filled HindIII site would regenerate the restriction site (andwas observed in several clones).

[0454] One liter cultures of each expression construct were grown, andfusion protein purified by affinity chromatography on either an amyloseresin column (pMAL constructs; resin supplied by New England Biolabs) orNi-chelate column (pET constructs; resin supplied by Qiagen or Novagen)as described [Williams et al. (1995), supra]. The integrity and purityof the fusion proteins were determined by Coomassie staining of SDS-PAGEprotein gels as well as Western blot analysis with either an affinitypurified goat polyclonal antiserum (Tech Lab), or a chicken polyclonalPEG prep, raised against the toxin B protein (CTB) as described above inExample 8. In both cases, affinity purification resulted in yields inexcess of 20 mg protein per liter culture, of which greater than 90% wasestimated to be full-length recombinant protein. It should be noted thatthe poly-histidine affinity tagged protein was released from the QiagenNi-NTA resin at low imidazole concentration (60 mM), necessitating theuse of a 40 mM imidazole rather than a 60 mM imidazole wash step duringpurification.

[0455] A periplasmically secreted version of pMB1750-2360 wasconstructed by replacement of the promoter and MBP coding region of thisconstruct with that from a related vector (pMAL™-p2; New EnglandBiolabs) in which a signal sequence is present at the N-terminus of theMBP, such that fusion protein is exported. This was accomplished bysubstituting a BglII-EcoRV promoter fragment from pMAL-p2 intopMB1750-2360. The yields of secreted, affinity purified protein(recovered from osmotic shock extracts as described by Riggs in CurrentProtocols in Molecular Biology, Vol. 2, Ausubel, et al., Eds. (1989),Current Protocols, pp. 16.6.1-16.6.14] from this vector (pMBp1750-2360)were 6.5 mg/liter culture, of which 50% was estimated to be full-lengthfusion protein.

[0456] The interval was also expressed in two non-overlapping fragments.pMB1750-1970 was constructed by introduction of a frameshift intopMB1750-2360, by restriction with HindIII, filling in the overhangingends and religation of the plasmid. Recombinant clones were selected byloss of the HindIII site, and further restriction map analysis.Recombinant protein expression from this vector was more than 20mg/liter of greater than 90% pure protein.

[0457] The complementary region was expressed in pMB1970-2360. Thisconstruct was created by removal of the 1750-1970 interval ofpMB1750-2360. This was accomplished by restriction of this plasmid withEcoRI (in the pMa1c polylinker 5′ to the insert) and III, filling in theoverhanging ends, and religation of the plasmid. The resultant plasmid,pMB1970-2360, was made using both intracellularly and secreted versionsof the pMB1750-2360 vector.

[0458] No fusion protein was secreted in the pMBp1970-2360 version,perhaps due to a conformational constraint that prevents export of thefusion protein. However, the intracellularly expressed vector producedgreater than 40 mg/liter of greater than 90% full-length fusion protein.

[0459] Constructs to precisely express the toxin B repeats in eitherpMa1c (pMB1850-2360) or pET16b (pPB1850-2360) were constructed asfollows. The DNA interval including the toxin B repeats was PCRamplified as described above utilizing PCR primers P14 (SEQ ID NO:19)and P8 (SEQ ID NO:14). Primer P14 adds a EcoRI site immediately flankingthe start of the toxin B repeats.

[0460] The amplified fragment was cloned into the pT7 Blue T-vector(Novagen) and recombinant clones in which single copies of the PCRfragment were inserted in either orientation were selected(pT71850-2360) and confirmed by restriction mapping. The insert wasexcised from two appropriately oriented independently isolatedpT71850-2360 plasmids, with EcoRI (5′ end of repeats) and PstI (in theflanking polylinker of the vector), and cloned into EcoRI/PstI cleavedpMa1c vector. The resulting construct (pMB1850-2360) was confirmed byrestriction analysis, and yielded 20 mg/l of soluble fusion protein[greater than 90% intact (i.e., nondegraded)] after affinitychromatography. Restriction of this plasmid with HindIII and religationof the vector resulted in the removal of the 1970-2360 interval. Theresultant construct (pMB1850-1970) expressed greater than 70 mg/liter of90% full length fusion protein.

[0461] The pPB1850-2360 construct was made by cloning a EcoRI (filledwith Klenow)-BamHI fragment from a pT71850-2360 vector (oppositeorientation to that used in the pMB1850-2360 construction) into NdeI(filled)/BamHI cleaved pET16b vector. Yields of affinity purifiedsoluble fusion protein were 15 mg/liter, of greater than 90% full lengthfusion protein.

[0462] Several smaller expression constructs from the 1750-2070 intervalwere also constructed in His-tagged pET vectors, but expression of theseplasmids in the BL21 (DE3) host resulted in the production of highlevels of mostly insoluble protein (see Table 23 and FIG. 19). Theseconstructs were made as follows. pPB1850-1970 was constructed by cloninga BglII-HindIII fragment of pPB1850-2360 into BglII/HindIII cleavedpET23b vector. pPB1850-2070 was constructed by cloning a BglII-PvuIIfragment of pPB1850-2360 into BglII/HincII cleaved pET23b vector.pPB1750-1970(c) was constructed by removal of the internal HindIIIfragment of a pPB1750-2360 vector in which the vector HindIII site wasregenerated during cloning (presumably by the addition of an A residueto the amplified PCR product by terminal transferase activity of Pfupolymerase). The pPB1750-1970(n) construct was made by insertion of theinsert containing the NdeI-HindIII fragment of pPB1750-2360 intoidentically cleaved pETHisb vector. All constructs were confirmed byrestriction digestion.

[0463] An expression construct that directs expression of the 10-470 aainterval of toxin B was constructed in the pMa1c vector as follows. ANheI (a site 5′ to the insert in the pET23 vector)-AfII (filled)fragment of the toxin B gene from pPB 10-1530 was cloned into XbaI(compatible with NheI)/HindIII (filled) pMa1c vector. The integrity ofthe construct (pMB10-470) was verified by restriction mapping and DNAsequencing of the 5′ clone junction using a MBP specific DNA primer (NewEngland Biolabs). However, all expressed protein was degraded to the MBPmonomer MW.

[0464] A second construct spanning this interval (aa 10-470) wasconstructed by cloning the PCR amplification product from a reactioncontaining the P9 (SEQ ID NO:15) and P10 (SEQ ID NO:16) primers (FIG.18) into the pETHisa vector. This was accomplished by cloning the PCRproduct as an EcoRI-blunt fragment into EcoRI-HincII restricted vectorDNA; recombinant clones were verified by restriction mapping. Althoughthis construct (pPB10-520) allowed expression and purification(utilizing the N-terminal polyhistidine affinity tag) of intact fusionprotein, yields were estimated at less than 500 μg per liter culture.

[0465] Higher yield of recombinant protein from this interval (aa10-520) were obtained by expression of the interval in two overlappingclones. The 10-330aa interval was cloned in both pETHisa and pMa1cvectors, using the BamHI-AfIII (filled) DNA fragment from pPB10-520.This fragment was cloned into BamHI-HindIII (filled) restricted pMa1c orBamHI-HincII restricted pETHisa vector. Recombinant clones were verifiedby restriction mapping. High level expression of either insoluble (pET)or soluble (pMa1) fusion protein was obtained. Total yields of fusionprotein from the pMB10-330 construct (FIG. 18) were 20 mg/liter culture,of which 10% was estimated to be full-length fusion protein. Althoughyields of this interval were higher and >90% full-length recombinantprotein produced when expressed from the pET construct, the pMa1 fusionwas utilized since the expressed protein was soluble and thus morelikely to have the native conformation.

[0466] The pMB260-520 clone was constructed by cloning EcoRI-XbaIcleaved amplified DNA from a PCR reaction containing the P11 (SEQ IDNO:17) and P10 (SEQ ID NO:16) DNA primers (FIG. 18) into similarlyrestricted pMa1c vector. Yields of affinity purified protein were 10mg/liter, of which approximately 50% was estimated to be full-lengthrecombinant protein.

[0467] The aa510-1110 interval was expressed as described below. Thisentire interval was expressed as a pMa1 fusion by cloning theNheI-HindIII fragment of pUCB10-1530 into XbaI-HindIII cleaved pMa1cvector. The integrity of the construct (pMB510-1110) was verified byrestriction mapping and DNA sequencing of the 5′ clone junction using aMBP specific DNA primer. The yield of affinity purified protein was 25mg/liter culture, of which 5% was estimated to be full-length fusionprotein (1 mg/liter).

[0468] To attempt to obtain higher yields, this region was expressed intwo fragments (aa510-820, and 820-1110) in the pMa1c vector. ThepMB510-820 clone was constructed by insertion of a SacI (in the pMa1cpolylinker 5′ to the insert)-HpaI DNA fragment from pMB510-1110 intoSacI/StuI restricted pMa1c vector. The pMB820-1110 vector wasconstructed by insertion of the HpaI-HindIII fragment of pUCB10-1530into BamHI (filled)/HindIII cleaved pMa1c vector. The integrity of theseconstructs were verified by restriction mapping and DNA sequencing ofthe 5′ clone junction using a MBP specific DNA primer.

[0469] Recombinant protein expressed from the pMB510-820 vector washighly unstable. However, high levels (20 mg/liter) of >90% full-lengthfusion protein were obtained from the pMB820-1105 construct. Thecombination of partially degraded pMB510-1110 protein (enriched for the510-820 interval) with the pMB820-1110 protein provides usable amountsof recombinant antigen from this interval.

[0470] The aa1100-1750 interval was expressed as described below. Theentire interval was expressed in the pMa1c vector from a construct inwhich the AccI(filled)-SpeI fragment of pPB10-1750 was inserted intoStuI/XbaI (XbaI is compatible with SpeI; StuI and filled AccI sites areboth blunt ended) restricted pMa1c. The integrity of this construct(pMB1100-1750) was verified by restriction mapping and DNA sequencing ofthe clone junction using a MBP specific DNA primer. Although 15 mg/literof affinity purified protein was isolated from cells harboring thisconstruct, the protein was greater than 99% degraded to MBP monomersize.

[0471] A smaller derivative of pMB1100-1750 was constructed byrestriction of pMB1100-1750 with AfII and SalI (in the pMa1c polylinker3′ to the insert), filling in the overhanging ends, and religating theplasmid. The resultant clone (verified by restriction digestion and DNAsequencing) has deleted the aa1530-1750 interval, was designatedpMB1100-1530. pMB1100-1530 expressed recombinant protein at a yield ofgreater than 40 mg/liter, of which 5% was estimated to be full-lengthfusion protein.

[0472] Three constructs were made to express the remaining interval.Initially, a BspHI (filled)-SpeI fragment from pPB10-1750 was clonedinto EcoRI(filled)/XbaI cleaved pMa1c vector. The integrity of thisconstruct (pMB1570-1750) was verified by restriction mapping and DNAsequencing of the 5′ clone junction using a MBP specific DNA primer.Expression of recombinant protein from this plasmid was very low,approximately 3 mg affinity purified protein per liter, and most wasdegraded to MBP monomer size. This region was subsequently expressedfrom a PCR amplified DNA fragment. A PCR reaction utilizing primers P13[SEQ ID NO:18; P13 was engineered to introduce an EcoRI site 5′ toamplified toxin B sequences] and P8 (SEQ ID NO: 14) was performed on C.difficile genomic DNA as described above. The amplified fragment wascleaved with EcoRI and SpeI, and cloned into EcoRI/XbaI cleaved pMa1cvector. The resultant clone (pMB1530-1750) was verified by restrictionmap analysis, and recombinant protein was expressed and purified. Theyield was greater than 20 mg protein per liter culture and it wasestimated that 25% was full-length fusion protein; this was asignificantly higher yield than the original pMB1570-1750 clone: Theinsert of pMB1530-1750 (in a EcoRI-SalI fragment) was transferred to thepETHisa vector (EcoRI/XhoI cleaved, XhoI and SalI ends are compatible).No detectable fusion protein was purified on Ni-Chelate columns fromsoluble lysates of cells induced to express fusion protein from thisconstruct. TABLE 23 Summary Of Toxin B Expression Constructs^(a) CloneAffinity Tag Yield (mg/liter) % Full Length pPB10-1750 none low(estimated from ? Western blot hyb) pPB10-1530 none low (as above) ?pMB10-470 MBP    15 mg/l 0% pPB10-520 poly-his   0.5 mg/l 20% pPB10-330poly-his  >20 mg/l (insoluble) 90% pMB10-330 MBP    20 mg/l 10%pMB260-520 MBP    10 mg/l 50% pMB510-1110 MBP    25 mg/l 5% pMB510-820MBP degraded (by Western blot hyb) pMB820-1110 MBP    20 mg/l 90%pMB1100-1750 MBP    15 mg/l 0% pMB1100-1530 MBP    40 mg/l 5%pMB1570-1750 MBP     3 mg/l <5% pPB1530-1750 poly-his no purifiedprotein ? detected pMB1530-1750 MBP    20 mg/l 25% pMB1750-2360 MBP  >20mg/l >90% pMBp1750-2360 MBP   6.5 mg/l (secreted) 50% pPB1750-2360poly-his  >20 mg/l >90% pMB1750-1970 MBP  >20 mg/l >90% pMB1970-2360 MBP   40 mg/l >90% pMBp1970-2360 MBP (no secretion) NA pMB1850-2360 MBP   20 mg/l >90% pPB1850-2360 poly-his    15 mg/l >90% pMB1850-1970 MBP   70 mg/l >90% pPB1850-1970 poly-his  >10 mg/l (insoluble) >90%pPB1850-2070 poly-his  >10 mg/l (insoluble) >90% pPB1750-1970(c)poly-his  >10 mg/l (insoluble) >90% pPB1750-1970(n) poly-his  >10 mg/l(insoluble) >90%

EXAMPLE 19 Identification, Purification and Induction of NeutralizingAntibodies Against Recombinant C. difficile Toxin B Protein

[0473] To determine whether recombinant toxin B polypeptide fragmentscan generate neutralizing antibodies, typically animals would first beimmunized with recombinant proteins and anti-recombinant antibodies aregenerated. These anti-recombinant protein antibodies are then tested forneutralizing ability in vivo or in vitro. Depending on the immunogenicnature of the recombinant polypeptide, the generation of high-titerantibodies against that protein may take several months. To acceleratethis process and identify which recombinant polypeptide(s) may be thebest candidate to generate neutralizing antibodies, depletion studieswere performed. Specifically, recombinant toxin B polypeptide werepre-screened by testing whether they have the ability to bind toprotective antibodies from a CTB antibody preparation and hence depletethose antibodies of their neutralizing capacity. Those recombinantpolypeptides found to bind CTB, were then utilized to generateneutralizing antibodies. This Example involved: a) identification ofrecombinant sub-regions within toxin B to which neutralizing antibodiesbind; b) identification of toxin B sub-region specific antibodies thatneutralize toxin B in vivo; and c) generation and evaluation ofantibodies reactive to recombinant toxin B polypeptides.

[0474] a) Identification of Recombinant Sub-Regions Within Toxin B towhich Neutralizing Antibodies Bind

[0475] Sub-regions within toxin B to which neutralizing antibodies bindwere identified by utilizing recombinant toxin B proteins to depleteprotective antibodies from a polyclonal pool of antibodies againstnative C. difficile toxin B. An in vivo assay was developed to evaluateprotein preparations for the ability to bind neutralizing antibodies.Recombinant proteins were first pre-mixed with antibodies directedagainst native toxin B (CTB antibody; see Example 8) and allowed toreact for one hour at 37° C. Subsequently, C. difficile toxin B (CTB;Tech Lab) was added at a concentration lethal to hamsters and incubatedfor another hour at 37° C. After incubation this mixture was injectedintraperitoneally (IP) into hamsters. If the recombinant polypeptidecontains neutralizing epitopes, the CTB antibodies will lose its abilityto protect the hamsters against death from CTB. If partial or completeprotection occurs with the CTB antibody-recombinant mixture, thatrecombinant contains only weak or non-neutralizing epitopes of toxin B.This assay was performed as follows.

[0476] Antibodies against CTB were generated in egg laying Leghorn hensas described in Example 8. The lethal dosage (LD₁₀₀) of C. difficiletoxin B when delivered I.P. into 40 g female Golden Syrian hamsters(Charles River) was determined to be 2.5 to 5 μg. Antibodies generatedagainst CTB and purified by PEG precipitation could completely protectthe hamsters at the I.P. dosage determined above. The minimal amount ofCTB antibody needed to afford good protection against 5 μg of CTB wheninjected I.P. into hamsters was also determined (1×PEG prep). Theseexperiments defined the parameters needed to test whether a givenrecombinant protein could deplete protective CTB antibodies.

[0477] The cloned regions tested for neutralizing ability cover theentire toxin B gene and were designated as Intervals (INT) 1 through 5(see FIG. 19). Approximately equivalent final concentrations of eachrecombinant polypeptide were tested. The following recombinantpolypeptides were used: 1) a mixture of intervals 1 and 2 (INT-1,2); 2)a mixture of Intervals 4 and 5 (INT-4, 5) and 3) Interval 3 (INT-3).Recombinant proteins (each at about 1 mg total protein) were firstpreincubated with a final CTB antibody concentration of 1× [i.e., pelletdissolved in original yolk volume as described in Example 1(c)] in afinal volume of 5 mls for 1 hour at 37° C. Twenty-five μg of CTB (at aconcentration of 5 μg/ml), enough CTB to kill 5 hamsters, was then addedand the mixture was then incubated for 1 hour at 37° C. Five, 40 gfemale hamsters (Charles River) in each treatment group were then eachgiven 1 ml of the mixture I.P. using a tuberculin syringe with a 27gauge needle. The results of this experiment are shown in Table 24.TABLE 24 Binding Of Neutralizing Antibodies By INT 3 Protein Number OfNumber Of Treatment Group¹ Animals Alive Animals Dead CTB antibodies 3 2CTB antibodies + INT1, 2 3 2 CTB antibodies + INT4, 5 3 2 CTBantibodies + INT 3 0 5

[0478] As shown in Table 24, the addition of recombinant proteins fromINT-1, 2 or INT-4, 5 had no effect on the in vivo protective ability ofthe CTB antibody preparation compared to the CTB antibody preparationalone. In contrast, INT-3 recombinant polypeptide was able to remove allof the toxin B neutralizing ability of the CTB antibodies asdemonstrated by the death of all the hamsters in that group.

[0479] The above experiment was repeated, using two smaller expressedfragments (pMB 1750-1970 and pMB 1970-2360, see FIG. 19) comprising theINT-3 domain to determine if that domain could be further subdividedinto smaller neutralizing epitopes. In addition, full-length INT-3polypeptide expressed as a nickel tagged protein (pPB1750-2360) wastested for neutralizing ability and compared to the original INT-3expressed MBP fusion (pMB1750-2360). The results are shown in Table 25.TABLE 25 Removal Of Neutralizing Antibodies By Repeat ContainingProteins Number Of Number Of Treatment Group¹ Animals Alive Animals DeadCTB antibodies 5 0 CTB antibodies + pPB1750-2360 0 5 CTB antibodies +pMB1750-2360 0 5 CTB antibodies + pMB1970-2360 3 2 CTB antibodies +pMB1750-1970 2 3

[0480] The results summarized in Table 25 indicate that the smallerpolypeptide fragments within the INT-3 domain, pMB1750-1970 andpMB1970-2360, partially lose the ability to bind to and removeneutralizing antibodies from the CTB antibody pool. These resultsdemonstrate that the full length INT-3 polypeptide is required tocompletely deplete the CTB antibody pool of neutralizing antibodies.This experiment also shows that the neutralization epitope of INT-3 canbe expressed in alternative vector systems and the results areindependent of the vector utilized or the accompanying fusion partner.

[0481] Other Interval 3 specific proteins were subsequently tested forthe ability to remove neutralizing antibodies within the CTB antibodypool as described above. The Interval 3 specific proteins used in thesestudies are summarized in FIG. 23. In FIG. 23 the followingabbreviations are used: pP refers to the pET23 vector; pM refers to thepMALc vector; B refers to toxin B; the numbers refer to the amino acidinterval expressed in the clone. The solid black ovals represent theMBP; and HHH represents the poly-histidine tag. Only recombinantproteins comprising the entire toxin B repeat domain (pMB1750-2360,pPB1750-2360 and pPB1850-2360) can bind and completely removeneutralizing antibodies from the CTB antibody pool. Recombinant proteinscomprising only a portion of the toxin B repeat domain were not capableof completely removing neutralizing antibodies from the CTB antibodypool (pMB1750-1970 and pMB1970-2360 could partially remove neutralizingantibodies while pMB1850-1970 and pPB1850-2070 failed to remove anyneutralizing antibodies from the CTB antibody pool).

[0482] The above results demonstrate that only the complete ligandbinding domain (repeat region) of the toxin B gene can bind andcompletely remove neutralizing antibodies from the CTB antibody pool.These results demonstrate that antibodies directed against the entiretoxin B repeat region are necessary for in vivo toxin neutralization(see FIG. 23; only the recombinant proteins expressed by thepMB1750-2360, pPB1750-2360 and pPB1850-2360 vectors are capable ofcompletely removing the neutralizing antibodies from the CTB antibodypool).

[0483] These results represent the first indication that the entirerepeat region of toxin B would be necessary for the generation ofantibodies capable of neutralizing toxin B, and that sub-regions may notbe sufficient to generate maximal titers of neutralizing antibodies.

[0484] b) Identification of Toxin B Sub-Region Specific Antibodies thatNeutralize Toxin B In Vivo

[0485] To determine if antibodies directed against the toxin B repeatregion are sufficient for neutralization, region specific antibodieswithin the CTB antibody preparation were affinity purified, and testedfor in vivo neutralization. Affinity columns containing recombinanttoxin B repeat proteins were made as described below. A separateaffinity column was prepared using each of the following recombinanttoxin B repeat proteins: pPB750-2360, pPB850-2360, pMB1750-1970 andpMB1970-2360.

[0486] For each affinity column to be made, four ml of PBS-washedActigel resin (Sterogene) was coupled overnight at room temperature with5-10 mg of affinity purified recombinant protein (first extensivelydialyzed into PBS) in 15 ml tubes (Falcon) containing a {fraction(1/10)} final volume Ald-coupling solution (1 M sodiumcyanoborohydride). Aliquots of the supernatants from the couplingreactions, before and after coupling, were assessed by Coomassiestaining of 7.5% SDS-PAGE gels. Based on protein band intensities, inall cases greater than 30% coupling efficiencies were estimated. Theresins were poured into 10 ml columns (BioRad), washed extensively withPBS, pre-eluted with 4M guanidine-HCl (in 10 mM Tris-HCl, pH 8.0) andreequilibrated in PBS. The columns were stored at 4° C.

[0487] Aliquots of a CTB IgY polyclonal antibody preparation (PEG prep)were affinity purified on each of the four columns as described below.The columns were hooked to a UV monitor (ISCO), washed with PBS and 40ml aliquots of a 2× PEG prep (filter sterilized using a 0.45μ filter)were applied. The columns were washed with PBS until the baseline valuewas re-established. The columns were then washed with BBStween to elutenonspecifically binding antibodies, and reequilibrated with PBS. Boundantibody was eluted from the column in 4M guanidine-HCl (in 10 nMTris-HCl, pH 8.0). The eluted antibody was immediately dialyzed againsta 100-fold excess of PBS at 4° C. for 2 hrs. The samples were thendialyzed extensively against at least 2 changes of PBS, and affinitypurified antibody was collected and stored at 4° C. The antibodypreparations were quantified by UV absorbance. The elution volumes werein the range of 4-8 ml. All affinity purified stocks contained similartotal antibody concentrations, ranging from 0.25-0.35% of the totalprotein applied to the columns.

[0488] The ability of the affinity purified antibody preparations toneutralize toxin B in vivo was determined using the assay outlined in a)above. Affinity purified antibody was diluted 1:1 in PBS before testing.The results are shown in Table 26.

[0489] In all cases similar levels of toxin neutralization was observed,such that lethality was delayed in all groups relative to preimmunecontrols. This result demonstrates that antibodies reactive to therepeat region of the toxin B gene are sufficient to neutralize toxin Bin vivo. The hamsters will eventually die in all groups, but this deathis maximally delayed with the CTB PEG prep antibodies. Thusneutralization with the affinity purified (AP) antibodies is not ascomplete as that observed with the CTB prep before affinitychromatography. This result may be due to loss of activity duringguanidine denaturation (during the elution of the antibodies from theaffinity column) or the presence of antibodies specific to other regionsof the toxin B gene that can contribute to toxin neutralization (presentin the CTB PEG prep). TABLE 26 Neutralization Of Toxin B By AffinityPurified Antibodies Number Number Animals Animals Treatment group^(a)Alive^(b) Dead^(b) Preimmune¹ 0 5 CTB¹; 400 μg 5 0 CTB (AP onpPB1750-2360);² 875 μg 5 0 CTB (AP on pMB1750-1970);² 875 μg 5 0 CTB (APon pMB1970-2360);² 500 μg 5 0 # received toxin premixed with antibodyraised against the indicated protein, as either: ¹4X antibody PEG prepor ²affinity purified (AP) antibody (from CTB PEG prep, on indicatedcolumns). The amount of specific antibody in each prep is indicated; theamount is directly determined for affinity purified preps and isestimated for the 4X # CTB as described in Example 15.

[0490] The observation that antibodies affinity purified against thenon-overlapping pMB1750-1970 and pMB1970-2360 proteins neutralized toxinB raised the possibility that either 1) antibodies specific to repeatsub-regions are sufficient to neutralize toxin B or 2) sub-regionspecific proteins can bind most or all repeat specific antibodiespresent in the. CTB polyclonal pool. This would likely be due toconformational similarities between repeats, since homology in theprimary amino acid sequences between different repeats is in the rangeof only 25-75% [Eichel-Streiber, et al. (1992) Molec. Gen. Genetics233:260]. These possibilities were tested by affinity chromatography.

[0491] The CTB PEG prep was sequentially depleted 2× on the pMB1750-1970column; only a small elution peak was observed after the secondchromatography, indicating that most reactive antibodies were removed.This interval depleted CTB preparation was then chromatographed on thepPB 1850-2360 column; no antibody bound to the column. The reactivity ofthe CTB and CTB (pMB1750-1970 depleted) preps to pPB1750-2360,pPB1850-2360, pMB1750-1970 and pMB1970-2360 proteins was then determinedby ELISA using the protocol described in Example 13(c). Briefly, 96-wellmicrotiter plates (Falcon, Pro-Bind Assay Plates) were coated withrecombinant protein by adding 100 μl volumes of protein at 1-2 μg/ml inPBS containing 0.005% thimerosal to each well and incubating overnightat 4° C. The next morning, the coating suspensions were decanted and thewells were washed three times using PBS. In order to block non-specificbinding sites, 100 μl of 1.0% BSA (Sigma) in PBS (blocking solution) wasthen added to each well, and the plates were incubated for 1 hr. at 37°C. The blocking solution was decanted and duplicate samples of 150 μl ofdiluted antibody was added to the first well of a dilution series. Theinitial testing serum dilution was ({fraction (1/200)} for CTB prep,(the concentration of depleted CTB was standardized by OD₂₈₀) inblocking solution containing 0.5% Tween 20, followed by 5-fold serialdilutions into this solution. This was accomplished by seriallytransferring 30 μl aliquots to 120 μl buffer, mixing, and repeating thedilution into a fresh well. After the final dilution, 30 μl was removedfrom the well such that all wells contained 120 μl final volume. A totalof 5 such dilutions were performed (4 wells total). The plates wereincubated for 1 hr at 37° C. Following this incubation, the seriallydiluted samples were decanted and the wells were washed three timesusing PBS containing 0.5% Tween 20 (PBST), followed by two 5 min washesusing BBS-Tween and a final three washes using PBST. To each well, 100μl of {fraction (1/1000)} diluted secondary antibody [rabbitanti-chicken IgG alkaline phosphatase (Sigma) diluted in blockingsolution containing 0.5% Tween 20] was added, and the plate wasincubated 1 hr at 37° C. The conjugate solutions were decanted and theplates were washed 6 times in PBST, then once in 50 mM Na₂CO₃, 10 mMMgCl₂, pH 9.5. The plates were developed by the addition of 100 μl of asolution containing 1 mg/ml para-nitro phenyl phosphate (Sigma)dissolved in 50 mM Na₂CO₃, 10 mM MgCl₂, pH 9.5 to each well. The plateswere then incubated at room temperature in the dark for 5-45 min. Theabsorbency of each well was measured at 410 nm using a Dynatech MR 700plate reader.

[0492] As predicted by the affinity chromatography results, depletion ofthe CTB prep on the pMB1750-1970 column removed all detectablereactivity to the pMB1970-2360 protein. The reciprocal purification of aCTB prep that was depleted on the pMB1970-2360 column yielded no boundantibody when chromatographed on the pMB1750-1970 column. These resultsdemonstrate that all repeat reactive antibodies in the CTB polyclonalpool recognize a conserved structure that is present in non-overlappingrepeats. Although it is possible that this conserved structurerepresents rare conserved linear epitopes, it appears more likely thatthe neutralizing antibodies recognize a specific protein conformation.This conclusion was also suggested by the results of Western blothybridization analysis of CTB reactivity to these recombinant proteins.

[0493] Western blots of 7.5% SDS-PAGE gels, loaded and electrophoresedwith defined quantities of each recombinant protein, were probed withthe CTB polyclonal antibody preparation. The blots were prepared anddeveloped with alkaline phosphatase as described in Example 3. Theresults are shown in FIG. 24.

[0494]FIG. 24 depicts a comparison of immunoreactivity of IgY antibodyraised against either native or recombinant toxin B antigen. Equalamounts of pMB1750-1970 (lane 1), pMB1970-2360 (lane 2), pPB1850-2360(lane 3) as well as a serial dilution of pPB1750-2360 (lanes 4-6comprising 1×, {fraction (1/10)}× and {fraction (1/100)} amounts,respectively) proteins were loaded in duplicate and resolved on a 7.5%SDS-PAGE gel. The gel was blotted and each half was hybridized with PEGprep IgY antibodies from chickens immunized with either native CTB orpPB1750-2360 protein. Note that the full-length pMB1750-1970 protein wasidentified only by antibodies reactive to the recombinant protein(arrows).

[0495] Although the CTB prep reacts with the pPB1750-2360, pPB1850-2360,and pMB1970-2360 proteins, no reactivity to the pMB1750-1970 protein wasobserved (FIG. 24). Given that all repeat reactive antibodies can bebound by this protein during affinity chromatography, this resultindicates that the protein cannot fold properly on Western blots. Sincethis eliminates all antibody reactivity, it is unlikely that the repeatreactive antibodies in the CTB prep recognize linear epitopes. This mayindicate that in order to induce protective antibodies, recombinanttoxin B protein will need to be properly folded. c) Generation andEvaluation of Antibodies Reactive To Recombinant Toxin B Polypeptides

[0496] i) Generation of Antibodies Reactive to Recombinant Toxin BProteins

[0497] Antibodies against recombinant proteins were generated in egglaying Leghorn hens as described in Example 13. Antibodies were raised[using Freunds adjuvant (Gibco) unless otherwise indicated] against thefollowing recombinant proteins: 1) a mixture of Interval 1+2 proteins(see FIG. 18); 2) a mixture of interval 4 and 5 proteins (see FIG. 18);3) pMB1970-2360 protein; 4) pPB1750-2360 protein; 5) pMB1750-2360; 6)pMB1750-2360 [Titermax adjuvant (Vaxcell)]; 7) pMB1750-2360 [Gerbuadjuvant (Biotech)]; 8) pMBp1750-2360 protein; 9) pPB1850-2360; and 10)pMB1850-2360.

[0498] Chickens were boosted at least 3 times with recombinant proteinuntil ELISA reactivity [using the protocol described in b) above withthe exception that the plates were coated with pPB1750-2360 protein] ofpolyclonal PEG preps was at least equal to that of the CTB polyclonalantibody PEG prep. ELISA titers were determined for the PEG preps fromall of the above immunogens and were found to be comparable ranging from1:12500 to 1:62500. High titers were achieved in all cases except in 6)pMB 1750-2360 in which strong titers were not observed using theTitermax adjuvant, and this preparation was not tested further.

[0499] ii) Evaluation of Antibodies Reactive to Recombinant Proteins byWestern Blot Hybridization

[0500] Western blots of 7.5% SDS-PAGE gels, loaded and electrophoresedwith defined quantities of recombinant protein (pMB1750-1970,pPB1850-2360, and pMB1970-2360 proteins and a serial dilution of thepPB1750-2360 to allow quantification of reactivity), were probed withthe CTB, pPB1750-2360, pMB1750-2360 and pMB1970-2360 polyclonal antibodypreparations (from chickens immunized using Freunds adjuvant). The blotswere prepared and developed with alkaline phosphatase as described abovein b).

[0501] As shown in FIG. 24, the CTB and pMB1970-2360 preps reactedstrongly with the pPB1750-2360, pPB1850-2360, and pMB1970-2360 proteinswhile the pPB1750-2360 and pMB1970-2360 (Gerbu) preparations reactedstrongly with all four proteins. The Western blot reactivity of thepPB1750-2360 and pMB1970-2360 (Gerbu) preparations were equivalent tothat of the CTB preparation, while reactivity of the pMB1970-2360preparation was <10% that of the CTB prep. Despite equivalent ELISAreactivities only weak reactivity (approximately 1%) to the recombinantproteins were observed in PEG preps from two independent groupsimmunized with the pMB1750-2360 protein and one group immunized with thepMB1750-2360 preparation using Freunds adjuvant.

[0502] Affinity purification was utilized to determine if thisdifference in immunoreactivity by Western blot analysis reflectsdiffering antibody titers. Fifty ml 2× PEG preparations from chickensimmunized with either pMB 1750-2360 or pMB1970-2360 protein werechromatographed on the pPB1750-2360 affinity column from b) above, asdescribed. The yield of affinity purified antibody (% total protein inpreparation) was equivalent to the yield obtained from a CTB PEGpreparation in b) above. Thus, differences in Western reactivity reflecta qualitative difference in the antibody pools, rather than quantitativedifferences., These results demonstrate that certain recombinantproteins are more effective at generating high affinity antibodies (asassayed by Western blot hybridization).

[0503] iii) In Vivo Neutralization of Toxin B Using Antibodies Reactiveto Recombinant Protein

[0504] The in vivo hamster model [described in Examples 9 and 14(b)] wasutilized to assess the neutralizing ability of antibodies raised againstrecombinant toxin B proteins. The results from three experiments areshown below in Tables 27-29.

[0505] The ability of each immunogen to neutralize toxin B in vivo hasbeen compiled and is shown in Table 30. As predicted from therecombinant protein-CTB premix studies (Table 24) only antibodies toInterval 3 (1750-2366) and not the other regions of toxin B (i.e.,intervals 1-5) are protective. Unexpectedly, antibodies generated toINT-3 region expressed in pMAL vector (pMB1750-2360 and pMB1750-2360)using Freunds adjuvant were non-neutralizing. This observation isreproducible, since no neutralization was observed in two independentimmunizations with pMB1750-2360 and one immunization with pMB1750-2360.The fact that 5× quantities of affinity purified toxin B repeat specificantibodies from pMB1750-2360 PEG preps cannot neutralize toxin B while1× quantities of affinity purified anti-CTB antibodies can (Table 28)demonstrates that the differential ability of CTB antibodies toneutralize toxin B is due to qualitative rather than quantitativedifferences in these antibody preparations. Only when this region wasexpressed in an alternative vector (pPB1750-2360) or using analternative adjuvant with the pMB1750-2360 protein were neutralizingantibodies generated. Importantly, antibodies raised using Freundsadjuvant to pPB1850-2360, which contains a fragment that is only 100amino acids smaller than recombinant pPB1750-2360, are unable toneutralize toxin B in vivo (Table 27); note also that the same vector isused for both pPB1850-2360 and pPB1750-2360. TABLE 27 In VivoNeutralization Of Toxin B Treatment Group^(a) Number Animals Alive^(b)Number Animals Dead^(b) Preimmune 0 5 CTB 5 0 INT1 + 2 0 5 INT 4 + 5 0 5pMB1750-2360 0 5 pMB1970-2360 0 5 pPB1750-2360 5 0

[0506] TABLE 28 In Vivo Neutralization Of Toxin B Using AffinityPurified Antibodies Number Animals Number Animals Treatment Group^(a)Alive^(b) Dead^(b) Preimmune(1) 0 5 CTB(1) 5 0 pPB1750-2360(1) 5 0 1.5mg anti-pMB1750-2360(2) 1 4 1.5 mg anti-pMB1970-2360(2) 0 5 300 μganti-CTB(2) 5 0 # either (1) 4X antibody PEG prep or (2) affinitypurified antibody (on a pPB1750-2360 resin), either 1.5 mg/group(anti-pMB1750-2360 and anti-pMB1970-2360; used undiluted affinitypurified antibody) or 350 μg/group (anti-CTB, repeat specific; used 1/5diluted anti-CTB antibody).

[0507] TABLE 29 Generation Of Neutralizing Antibodies Utilizing TheGerbu Adjuvant Number Animals Number Animals Treatment Group^(a)Alive^(b) Dead^(b) Preimmune 0 5 CTB 5 0 pMB1970-2360 0 5 pMB1850-2360 05 pPB1850-2360 0 5 pMB1750-2360 (Gerbu adj) 5 0

[0508] TABLE 30 In Vivo Neutralization Of Toxin B Antigen In vivo TestedUtilized Neutral- Immunogen Adjuvant Preparation^(a) For AP ization^(b)Preimmune NA¹ PEG NA no CTB (native) Titermax PEG NA yes CTB (native)Titermax AP pPB1750-2360 yes CTB (native) Titermax AP pPB1850-2360 yesCTB (native) Titermax AP pPB1750-1970 yes CTB (native) Titermax APpPB1970-2360 yes pMB1750-2360 Freunds PEG NA no pMB1750-2360 Freunds APpPB1750-2360 no pMB1750-2360 Gerbu PEG NA yes pMB1970-2360 Freunds PEGNA no pMB1970-2360 Freunds AP pPB1750-2360 no pPB1750-2360 Freunds PEGNA yes pPB1850-2360 Freunds PEG NA no pMB1850-2360 Freunds PEG NA no INT1 + 2 Freunds PEG NA no INT 4 + 5 Freunds PEG NA no

[0509] The pPB1750-2360 antibody pool confers significant in vivoprotection, equivalent to that obtained with the affinity purified CTBantibodies. This correlates with the observed high affinity of thisantibody pool (relative to the pMB 1750-2360 or pMB1970-2360 pools) asassayed by Western blot analysis (FIG. 24). These results provide thefirst demonstration that in vivo neutralizing antibodies can be inducedusing recombinant toxin B protein as immunogen.

[0510] The failure of high concentrations of antibodies raised againstthe pMB1750-2360 protein (using Freunds adjuvant) to neutralize, whilethe use of Gerbu adjuvant and pMB1750-2360 protein generates aneutralizing response, demonstrates that conformation or presentation ofthis protein is essential for the induction of neutralizing antibodies.These results are consistent with the observation that the neutralizingantibodies produced when native CTB is used as an immunogen appear torecognize conformational epitopes [see section b) above]. This is thefirst demonstration that the conformation or presentation of recombinanttoxin B protein is essential to generate high titers of neutralizingantibodies.

EXAMPLE 20 Determination of Quantitative and Qualitative DifferencesBetween pMB1750-2360, pMB1750-2360 (Gerbu) or pPB1750-2360 IgYPolyclonal Antibody Preparations

[0511] In Example 19, it was demonstrated that toxin B neutralizingantibodies could be generated using specific recombinant toxin Bproteins (pPB1750-2360) or specific adjuvants. Antibodies raised againstpMB1750-2360 were capable of neutralizing the enterotoxin effect oftoxin B when the recombinant protein was used to immunize hens inconjunction with the Gerbu adjuvant, but not when Freunds adjuvant wasused. To determine the basis for these antigen and adjuvantrestrictions, toxin B-specific antibodies present in the neutralizingand non-neutralizing PEG preparations were isolated by affinitychromatography and tested for qualitative or quantitative differences.The example involved a) purification of anti-toxin B specific antibodiesfrom pMB1750-2360 and pPB1750-2360 PEG preparations and b) in vivoneutralization of toxin B using the affinity purified antibody.

[0512] a) Purification of Specific Antibodies from pMB1750-2360 andpPB1750-2360 PEG Preparations

[0513] To purify and determine the concentration of specific antibodies(expressed as the percent of total antibody) within the pPB1750-2360(Freunds and Gerbu) and pPB1750-2360 PEG preparations, definedquantities of these antibody preparations were chromatographed on anaffinity column containing the entire toxin B repeat region (pPB1750-2360). The amount of affinity purified antibody was thenquantified.

[0514] An affinity column containing the recombinant toxin B repeatprotein, pPB1750-2360, was made as follows. Four ml of PBS-washedActigel resin (Sterogene) was coupled with 5 mg of pPB1750-2360 affinitypurified protein (dialyzed into PBS; estimated to be greater than 95%full length fusion protein) in a 15 ml tube (Falcon) containing{fraction (1/10)} final volume Aid-coupling solution (1M sodiumcyanoborohydride). Aliquots of the supernatant from the couplingreactions, before and after coupling, were assessed by Coomassiestaining of 7.5% SDS-PAGE gels. Based on protein band intensities,greater than 95% (approximately 5 mg) of recombinant protein was coupledto the resin. The coupled resin was poured into a 10 ml column (BioRad),washed extensively with PBS, pre-eluted with 4M guanidine-HCl (in 10 mMTris-HCl, pH 8.0; 0.005% thimerosal) and re-equilibrated in PBS andstored at 4° C.

[0515] Aliquots of pMB1750-2360, pMB1750-2360 (Gerbu) or pPB1750-2360IgY polyclonal antibody preparations (PEG preps) were affinity purifiedon the above column as follows. The column was attached to an UV monitor(ISCO), and washed with PBS. Forty ml aliquots of 2×PEG preps (filtersterilized using a 0.45μ filter and quantified by OD₂₈₀ beforechromatography) was applied. The column was washed with PBS until thebaseline was re-established (the column flow-through was saved), washedwith BBSTween to elute nonspecifically binding antibodies andre-equilibrated with PBS. Bound antibody was eluted from the column in4M guanidine-HCl (in 10 mM Tris-HCL, pH 8.0, 0.005% thimerosal) and theentire elution peak collected in a 15 ml tube (Falcon). The column wasre-equilibrated, and the column eluate re-chromatographed as describedabove. The antibody preparations were quantified by UV absorbance (theelution buffer was used to zero the spectrophotometer). Approximately 10fold higher concentrations of total purified antibody was obtained uponelution of the first chromatography pass relative to the second pass.The low yield from the second chromatography pass indicated that most ofthe specific antibodies were removed by the first round ofchromatography.

[0516] Pools of affinity purified specific antibodies were prepared bydialysis of the column elutes after the first column chromatography passfor the pMB1750-2360, pMB1750-2360 (Gerbu) or pPB1750-2360 IgYpolyclonal antibody preparations. The elutes were collected on ice andimmediately dialyzed against a 100-fold volume of PBS at 4° C. for 2hrs. The samples were then dialyzed against 3 changes of a 65-foldvolume of PBS at 4° C. Dialysis was performed for a minimum of 8 hrs perchange of PBS. The dialyzed samples were collected, centrifuged toremove insoluble debris, quantified by OD₂₈₀, and stored at 4° C.

[0517] The percentage of toxin B repeat-specific antibodies present ineach preparation was determined using the quantifications of antibodyyields from the first column pass (amount of specific antibody recoveredafter first pass/total protein loaded). The yield of repeat-specificaffinity purified antibody (expressed as the percent of total protein inthe preparation) in: 1) the pMB1750-2360 PEG prep was approximately0.5%, 2) the pMB1750-2360 (Gerbu) prep was approximately 2.3%, and 3)the pPB1750-2360 prep was approximately 0.4%. Purification of a CTB IgYpolyclonal antibody preparation on the same column demonstrated that theconcentration of toxin B repeat specific antibodies in the CTBpreparation was 0.35%.

[0518] These results demonstrate that 1) the use of Gerbu adjuvantenhanced the titer of specific antibody produced against thepMB1750-2360 protein 5-fold relative to immunization using Freundsadjuvant, and 2) the differences seen in the in vivo neutralizationability of the pMB1750-2360 (not neutralizing) and pPB1750-2360(neutralizing) and CTB (neutralizing) PEG preps seen in Example 19 wasnot due to differences in the titers of repeat-specific antibodies inthe three preparations because the titer of repeat-specific antibody wassimilar for all three preps; therefore the differing ability of thethree antibody preparations to neutralize toxin B must reflectqualitative differences in the induced toxin B repeat-specificantibodies. To confirm that qualitative differences exist betweenantibodies raised in hens immunized with different recombinant proteinsand/or different adjuvants, the same amount of affinity purifiedanti-toxin B repeat (aa 1870-2360 of toxin B) antibodies from thedifferent preparations was administered to hamsters using the in vivohamster model as described below.

[0519] b) In Vivo Neutralization of Toxin B Using Affinity PurifiedAntibody

[0520] The in vivo hamster model was utilized to assess the neutralizingability of the affinity purified antibodies raised against recombinanttoxin B proteins purified in (a) above. As well, a 4× IgY PEGpreparation from a second independent immunization utilizing thepPB1750-2360 antigen with Freunds adjuvant was tested for in vivoneutralization. The results are shown in Table 31.

[0521] The results shown in Table 31 demonstrate that:

[0522] 1) as shown in Example 19 and reproduced here, 1.5 mg of affinitypurified antibody from pMB1750-2360 immunized hens using Freundsadjuvant does not neutralize toxin B in vivo. However, 300 μg ofaffinity purified antibody from similarly immunized hens utilizing Gerbuadjuvant demonstrated complete neutralization of toxin B in vivo. Thisdemonstrates that Gerbu adjuvant, in addition to enhancing the titer ofantibodies reactive to the pMB1750-2360 antigen relative to Freundsadjuvant (demonstrated in (a) above), also enhances the yield ofneutralizing antibodies to this antigen, greater than 5 fold.

[0523] 2) Complete in vivo neutralization of toxin B was observed with1.5 mg of affinity purified antibody from hens immunized withpPB1750-2360 antigen, but not with pMB1750-2360 antigen, when Freundsadjuvant was used. This demonstrates, using standardized toxin Brepeat-specific antibody concentrations, that neutralizing antibodieswere induced when pPB1750-2360 but not pMB1750-2360 was used as theantigen with Freunds adjuvant.

[0524] 3) Complete in vivo neutralization was observed with 300 μg ofpMB1750-2360 (Gerbu) antibody, but not with 300 μg of pPB1750-2360(Freunds) antibody. Thus the pMB1750-2360 (Gerbu) antibody has a highertiter of neutralizing antibodies than the pPB1750-2360 (Freunds)antibody.

[0525] 4) Complete neutralization of toxin B was observed using 300 μgof CTB antibody [affinity purified (AP)] but not 100 μg CTB antibody (APor PEG prep). This demonstrates that greater than 100 μg of toxin Brepeat-specific antibody (anti-CTB) is necessary to neutralize 25 μgtoxin B in vivo in this assay, and that affinity purified antibodiesspecific to the toxin B repeat interval neutralize toxin B aseffectively as the PEP prep of IgY raised against the entire CTB protein(shown in this assay).

[0526] 5) As was observed with the initial pPB1750-2360 (IgY) PEGpreparation (Example 19), complete neutralization was observed with aIgY PEG preparation isolated from a second independent group ofpPB1750-2360 (Freunds) immunized hens. This demonstrates thatneutralizing antibodies are reproducibly produced when hens areimmunized with pPB1750-2360 protein utilizing Freunds adjuvant. TABLE 31In vivo Neutralization Of Toxin B Using Affinity Purified AntibodiesNumber Animals Number Animals Treatment Group^(a) Alive^(b) Dead^(b)Preimmune¹ 0 5 CTB (300 μg)² 5 0 CTB (100 μg)² 1 4 pMB1750-2360 (G) (5mg)² 5 0 pMB1750-2360 (G) (1.5 mg)² 5 0 pMB1750-2360 (G) (300 μg)² 5 0pMB1750-2360 (F) (1.5 mg)² 0 5 pPB1750-2360 (F) (l.5 mg)² 5 0pPB1750-2360 (F) (300 μg)² 1 4 CTB (100 μg)³ 2 3 pPB1750-2360 (F) (500μg)¹ 5 0 # mix injected per hamster). Each treatment group receivedtoxin premixed with antibody raised against the indicated protein (G =gerbu adjuvant, F = Freunds adjuvant).

EXAMPLE 21 Diagnostic Enzyme Immunoassays for C. difficile Toxins A andB

[0527] The ability of the recombinant toxin proteins and antibodiesraised against these recombinant proteins (described in the aboveexamples) to form the basis of diagnostic assays for the detection ofclostridial toxin in a sample was examined. Two immunoassay formats weretested to quantitatively detect C. difficile toxin A and toxin B from abiological specimen. The first format involved a competitive assay inwhich a fixed amount of recombinant toxin A or B was immobilized on asolid support (e.g., microtiter plate wells) followed by the addition ofa toxin-containing biological specimen mixed with affinity-purified orPEG fractionated antibodies against recombinant toxin A or B. If toxinis present in a specimen, this toxin will compete with the immobilizedrecombinant toxin protein for binding to the anti-recombinant antibodythereby reducing the signal obtained following the addition of areporter reagent. The reporter reagent detects the presence of antibodybound to the immobilized toxin protein.

[0528] In the second format, a sandwich immunoassay was developed usingaffinity-purified antibodies to recombinant toxin A and B. Theaffinity-purified antibodies to recombinant toxin A and B were used tocoat microtiter wells instead of the recombinant polypeptides (as wasdone in the competitive assay format). Biological samples containingtoxin A or B were then added to the wells followed by the addition of areporter reagent to detect the presence of bound toxin in the well.

[0529] a) Competitive Immunoassay for the Detection of C. difficileToxin

[0530] Recombinant toxin A or B was attached to a solid support bycoating 96 well microtiter plates with the toxin protein at aconcentration of 1 μg/ml in PBS. The plates were incubated overnight at2-8° C. The following morning, the coating solutions were removed andthe remaining protein binding sites on the wells were blocked by fillingeach well with a PBS solution containing 0.5% BSA and 0.05% Tween-20.Native C. difficile toxin A or B (Tech Lab) was diluted to 4 μg/ml instool extracts from healthy Syrian hamsters (Sasco). The stool extractswere made by placing fecal pellets in a 15 ml centrifuge tube; PBS wasadded at 2 ml/pellet and the tube was vortexed to create a uniformsuspension. The tube was then centrifuged at 2000 rpm for 5 min at roomtemperature. The supernatant was removed; this comprises the stoolextract. Fifty μl of the hamster stool extract was pipetted into eachwell of the microtiter plates to serve as the diluent for serialdilutions of the 4 μg/ml toxin samples. One hundred μl of the toxinsamples at 4 μg/ml was pipetted into the first row of wells in themicrotiter plate, and 50 μl aliquots were removed and diluted seriallydown the plate in duplicate. An equal volume of affinity purifiedanti-recombinant toxin antibodies [1 ng/well of anti-pMA1870-2680antibody was used for the detection of toxin A; 0.5 ng/well ofanti-pMB1750-2360(Gerbu) was used for the detection of toxin B] wereadded to appropriate wells, and the plates were incubated at roomtemperature for 2 hours with gentle agitation. Wells serving as negativecontrol contained antibody but no native toxin to compete for binding.

[0531] Unbound toxin and antibody were removed by washing the plates 3to 5 times with PBS containing 0.05% Tween-20. Following the wash step,100 μl of rabbit anti-chicken IgG antibody conjugated to alkalinephosphatase (Sigma) was added to each well and the plates were incubatedfor 2 hours at room temperature. The plates were then washed as beforeto remove unbound secondary antibody. Freshly prepared alkalinephosphatase substrate (1 mg/ml p-nitrophenyl phosphate (Sigma) in 50 mMNa₂CO₃, pH 9.5; 10 mM MgCl₂) was added to each well. Once sufficientcolor developed, the plates were read on a Dynatech MR700 microtiterplate reader using a 410 nm filter.

[0532] The results are summarized in Tables 32 and 33. For the resultsshown in Table 32, the wells were coated with recombinant toxin Aprotein (pMA1870-2680). The amount of native toxin A added (present asan addition to solubilized hamster stool) to a given well is indicated(0 to 200 ng). Antibody raised against the recombinant toxin A protein,pMA1870-2680, was affinity purified on the an affinity column containingpPA1870-2680 (described in Example 20). As shown in Table 32, therecombinant toxin A protein and affinity-purified antitoxin can be usedfor the basis of a competitive immunoassay for the detection of toxin Ain biological samples.

[0533] Similar results were obtained using the recombinant toxin B, pPB1750-2360, and antibodies raised against pMB1750-2360(Gerbu). For theresults shown in Table 33, the wells were coated with recombinant toxinB protein (pPB1750-2360). The amount of native toxin B added (present asan addition to solubilized hamster stool) to a given well is indicated(0 to 200 ng). Antibody raised against the recombinant toxin B protein,pMB1750-2360(Gerbu), was affinity purified on the an affinity columncontaining pPB 1850-2360 (described in Example 20). As shown in Table33, the recombinant toxin B protein and affinity-purified antitoxin canbe used for the basis of a competitive immunoassay for the detection oftoxin B in biological samples.

[0534] In this competition assay, the reduction is consideredsignificant over the background levels at all points; therefore theassay can be used to detect samples containing less than 12.5 ng toxinA/well and as little as 50-100 ng toxin B/well. TABLE 32 CompetitiveInhibition Of Anti-C. difficile Toxin A By Native Toxin A ng ToxinA/Well OD₄₁₀ Readout 200 0.176 100 0.253 50 0.240 25 0.259 12.5 0.3096.25 0.367 3.125 0.417 0 0.590

[0535] TABLE 33 Competitive Inhibition Of Anti-C. difficile Toxin B ByNative Toxin B ng Toxin B/Well OD₄₁₀ Readout 200 0.392 100 0.566 500.607 25 0.778 12.5 0.970 6.25 0.902 3.125 1.040 0 1.055

[0536] These competitive inhibition assays demonstrate that native C.difficile toxins and recombinant C. difficile toxin proteins can competefor binding to antibodies raised against recombinant C. difficile toxinsdemonstrating that these anti-recombinant toxin antibodies provideeffective diagnostic reagents.

[0537] b) Sandwich Immunoassay for the Detection of C. difficile Toxin

[0538] Affinity-purified antibodies against recombinant toxin A or toxinB were immobilized to 96 well microtiter plates as follows. The wellswere passively coated overnight at 4° C. with affinity purifiedantibodies raised against either pMA1870-2680 (toxin A) orpMB1750-2360(Gerbu) (toxin B). The antibodies were affinity purified asdescribed in Example 20. The antibodies were used at a concentration of1 μg/ml and 100 μl was added to each microtiter well. The wells werethen blocked with 200 μl of 0.5% BSA in PBS for 2 hours at roomtemperature and the blocking solution was then decanted. Stool samplesfrom healthy Syrian hamsters were resuspended in PBS, pH 7.4 (2 mlPBS/stool pellet was used to resuspend the pellets and the sample wascentrifuged as described above). The stool suspension was then spikedwith native C. difficile toxin A or B (Tech Lab) at 4 μg/ml. The stoolsuspensions containing toxin (either toxin A or toxin B) were thenserially diluted two-fold in stool suspension without toxin and 50 μlwas added in duplicate to the coated microtiter wells. Wells containingstool suspension without toxin served as the negative control.

[0539] The plates were incubated for 2 hours at room temperature andthen were washed three times with PBS. One hundred μl of either goatanti-native toxin A or goat anti-native toxin B (Tech Lab) diluted1:1000 in PBS containing 1% BSA and 0.05% Tween 20 was added to eachwell. The plates were incubated for another 2 hours at room temperature.The plates were then washed as before and 100 μl of alkalinephosphatase-conjugated rabbit anti-goat IgG (Cappel, Durham, N.C.) wasadded at a dilution of 1:1000. The plates were incubated for another 2hours at room temperature. The plates were washed as before thendeveloped by the addition of 100 μl/well of a substrate solutioncontaining 1 mg/ml p-nitrophenyl phosphate (Sigma) in 50 mM Na₂CO₃, pH9.5; 10 mM MgCl₂. The absorbance of each well was measured using a platereader (Dynatech) at 410 nm. The assay results are shown in Tables 34and 35. TABLE 34 C. difficile Toxin A Detection In Stool Using Affinity-Purified Antibodies Against Toxin A ng Toxin A/Well OD₄₁₀ Readout 2000.9 100 0.8 50 0.73 25 0.71 12.5 0.59 6.25 0.421 0 0

[0540] TABLE 35 C. difficile Toxin B Detection In Stool Using Affinity-Purified Antibodies Against Toxin B ng Toxin B/Well OD₄₁₀ Readout 2001.2 100 0.973 50 0.887 25 0.846 12.5 0.651 6.25 0.431 0 0.004

[0541] The results shown in Tables 34 and 35 show that antibodies raisedagainst recombinant toxin A and toxin B fragments can be used to detectthe presence of C. difficile toxin in stool samples. These antibodiesform the basis for a sensitive sandwich immunoassay which is capable ofdetecting as little as 6.25 ng of either toxin A or B in a 50 μl stoolsample. As shown above in Tables 34 and 35, the background for thissandwich immunoassay is extremely low; therefore, the sensitivity ofthis assay is much lower than 6.25 ng toxin/well. It is likely thattoxin levels of 0.5 to 1.0 μg/well could be detected by this assay.

[0542] The results shown above in Tables 32-35 demonstrate clear utilityof the recombinant reagents in C. difficile toxin detection systems.

EXAMPLE 22 Construction and Expression of C. botulinum C Fragment FusionProteins

[0543] The C. botulinum type A neurotoxin gene has been cloned andsequenced [Thompson, et al., Eur. J. Biochem. 189:73 (1990)]. Thenucleotide sequence of the toxin gene is available from the EMBL/GenBanksequence data banks under the accession number X52066; the nucleotidesequence of the coding region is listed in SEQ ID NO:27. The amino acidsequence of the C. botulinum type A neurotoxin is listed in SEQ IDNO:28. The type A neurotoxin gene is synthesized as a single polypeptidechain which is processed to form a dimer composed of a light and a heavychain linked via disulfide bonds. The 50 kD carboxy-terminal portion ofthe heavy chain is referred to as the C fragment or the H_(C) domain.

[0544] Previous attempts by others to express polypeptides comprisingthe C fragment of C. botulinum type A toxin as a native polypeptide(e.g., not as a fusion protein) in E. coli have been unsuccessful [H.F.LaPenotiere, et al. in Botulinum and Tetanus Neurotoxins, DasGupta, Ed.,Plenum Press, New York (1993), pp. 463-466]. Expression of the Cfragment as a fusion with the E. coli MBP was reported to result in theproduction of insoluble protein (H. F. LaPenotiere, et al., supra).

[0545] In order to produce soluble recombinant C fragment proteins in E.coli, fusion proteins comprising a synthetic C fragment gene derivedfrom the C. botulinum type A toxin and either a portion of the C.difficile toxin protein or the MBP were constructed. This exampleinvolved a) the construction of plasmids encoding C fragment fusionproteins and b) expression of C. botulinum C fragment fusion proteins inE. coli.

[0546] a) Construction of Plasmids Encoding C Fragment Fusion Proteins

[0547] In Example 11, it was demonstrated that the C. difficile toxin Arepeat domain can be efficiently expressed and purified in E. coli aseither native (expressed in the pET 23a vector in clone pPA1870-2680) orfusion (expressed in the pMALc vector as a fusion with the E. coli MBPin clone pMA1870-2680) proteins. Fusion proteins comprising a fusionbetween the MBP, portions of the C. difficile toxin A repeat domain(shown to be expressed as a soluble fusion protein) and the C fragmentof the C. botulinum type A toxin were constructed. A fusion proteincomprising the C fragment of the C. botulinum type A toxin and the MBPwas also constructed.

[0548]FIG. 25 provides a schematic representation of the botulinalfusion proteins along with the donor constructs containing the C.difficile toxin A sequences or C. botulinum C fragment sequences whichwere used to generate the botulinal fusion proteins. In FIG. 25, thesolid boxes represent C. difficile toxin A gene sequences, the openboxes represent C. botulinum C fragment sequences and the solid blackovals represent the E. coli MBP. When the name for a restriction enzymeappears inside parenthesis, this indicates that the restriction site wasdestroyed during construction. An asterisk appearing with the name for arestriction enzyme indicates that this restriction site was recreated atthe cloning junction.

[0549] In FIG. 25, a restriction map of the pMA1870-2680 andpPA1100-2680 constructs (described in Example 11) which containsequences derived from the C. difficile toxin A repeat domain are shown;these constructs were used as the source of C. difficile toxin A genesequences for the construction of plasmids encoding fusions between theC. botulinum C fragment gene and the C. difficile toxin A gene. ThepMA1870-2680 expression construct expresses high levels of soluble,intact fusion protein (20 mg/liter culture) which can be affinitypurified on an amylose column (purification described in Example 11 d).

[0550] The pAlterBot construct (FIG. 25) was used as the source of C.botulinum C fragment gene sequences for the botulinal fusion proteins.pAlterBot was obtained from J. Middlebrook and R. Lemley at the U.S.Department of Defense. pAlterBot contains a synthetic C. botulinum Cfragment inserted in to the pALTER-1® vector (Promega). This synthetic Cfragment gene encodes the same amino acids as does the naturallyoccurring C fragment gene. The naturally occurring C fragment sequences,like most clostridial genes, are extremely A/T rich (Thompson et al.,supra). This high A/T content creates expression difficulties in E. coliand yeast due to altered codon usage frequency and fortuitouspolyadenylation sites, respectively. In order to improve the expressionof C fragment proteins in E. coli, a synthetic version of the gene wascreated in which the non-preferred codons were replaced with preferredcodons.

[0551] The nucleotide sequence of the C. botulinum C fragment genesequences contained within pAlterBot is listed in SEQ ID NO:22. Thefirst six nucleotides (ATGGCT) encode a methionine and alanine residue,respectively. These two amino acids result from the insertion of the C.botulinum C fragment sequences into the pALTER® vector and provide theinitiator methionine residue. The amino acid sequence of the C.botulinum C fragment encoded by the sequences contained within pAlterBotis listed in SEQ ID NO:23. The first two amino acids (Met Ala) areencoded by vector-derived sequences. From the third amino acid residueonward (Arg), the amino acid sequence is identical to that found in theC. botulinum type A toxin gene.

[0552] The pMA1870-2680, pPA1100-2680 and pAlterBot constructs were usedas progenitor plasmids to make expression constructs in which fragmentsof the C. difficile toxin A repeat domain were expressed as geneticfusions with the C. botulinum C fragment gene using the pMAL-cexpression vector (New England BioLabs). The pMAL-c expression vectorgenerates fusion proteins which contain the MBP at the amino-terminalend of the protein. A construct, pMBot, in which the C. botulinum Cfragment gene was expressed as a fusion with only the MBP wasconstructed (FIG. 25). Fusion protein expression was induced from E.coli strains harboring the above plasmids, and induced protein wasaffinity purified on an amylose resin column.

[0553] i) Construction of pBlueBot

[0554] In order to facilitate the cloning of the C. botulinum C fragmentgene sequences into a number of desired constructs, the botulinal genesequences were removed from pAlterBot and were inserted into thepBluescript plasmid (Stratagene) to generate pBlueBot (FIG. 25).pBlueBot was constructed as follows. Bacteria containing the pAlterBotplasmid were grown in medium containing tetracycline and plasmid DNA wasisolated using the QIAprep-spin Plasmid Kit (Qiagen). One microgram ofpAlterBot DNA was digested with NcoI and the resulting 3′ recessedsticky end was made blunt using the Klenow fragment of DNA polymerase I(here after the Klenow fragment). The pAlterBot DNA was then digestedwith HindIII to release the botulinal gene sequences (the Bot insert) asa blunt (filled NcoI site)-HindIII fragment. pBluescript vector DNA wasprepared by digesting 200 ng of pBluescript DNA with SmaI and HindIII.The digestion products from both plasmids were resolved on an agarosegel. The appropriate fragments were removed from the gel, mixed andpurified utilizing the Prep-a-Gene kit (BioRad). The eluted DNA was thenligated using T4 DNA ligase and used to transform competent DH5α cells(Gibco-BRL). Host cells were made competent for transformation using thecalcium chloride protocol of Sanbrook et al., supra at 1.82-1.83.Recombinant clones were isolated and confirmed by restriction digestionusing standard recombinant molecular biology techniques (Sambrook et al,supra). The resultant clone, pBlueBot, contains several useful uniquerestriction sites flanking the Bot insert (i.e., the C. botulinum Cfragment sequences derived from pAlterBot) as shown in FIG. 25.

[0555] ii) Construction Of C. difficile/C. botulinum/MBP Fusion Proteins

[0556] Constructs encoding fusions between the C. difficile toxin A geneand the C. botulinum C fragment gene and the MBP were made utilizing thesame recombinant DNA methodology outlined above; these fusion proteinscontained varying amounts of the C. difficile toxin A repeat domain.

[0557] The pMABot clone contains a 2.4 kb insert derived from the C.difficile toxin A gene fused to the Bot insert (i.e, the C. botulinum Cfragment sequences derived from pAlterBot). pMABot (FIG. 25) wasconstructed by mixing gel-purified DNA from NotI/HindIII digestedpBlueBot (the 1.2 kb Bot fragment), SpeI/NotI digested pPA1100-2680 (the2.4 kb C. difficile toxin A repeat fragment) and XbaI/HindIII digestedpMAL-c vector. Recombinant clones were isolated, confirmed byrestriction digestion and purified using the QIAprep-spin Plasmid Kit(Qiagen). This clone expresses the toxin A repeats and the botulinal Cfragment protein sequences as an in-frame fusion with the MBP.

[0558] The pMCABot construct contains a 1.0 kb insert derived from theC. difficile toxin A gene fused to the Bot insert (i.e, the C. botulinumC fragment sequences derived from pAlterBot). pMCABot was constructed bydigesting the pMABot clone with EcoRI to remove the 5′ end of the C.difficile toxin A repeat (see FIG. 25, the pMAL-c vector contains aEcoRI site 5′ to the C. difficile insert in the pMABot clone). Therestriction sites were filled and religated together after gelpurification. The resultant clone (pMCABot, FIG. 25) generated anin-frame fusion between the MBP and the remaining 3′ portion of the C.difficile toxin A repeat domain fused to the Bot gene.

[0559] The pMNABot clone contains the 1 kb SpeI/EcoRI (filled) fragmentfrom the C. difficile toxin A repeat domain (derived from clone pPA1100-2680) and the 1.2 kb C. botulinum C fragment gene as a NcoI(filled)/HindIII fragment (derived from pAlterBot). These two fragmentswere inserted into the pMAL-c vector digested with XbaI/HindIII. The twoinsert fragments were generated by digestion of the appropriate plasmidwith EcoRI (pPA1100-2680) or NcoI (pAlterBot) followed by treatment withthe Klenow fragment. After treatment with the Klenow fragment, theplasmids were digested with the second enzyme (either SpeI or HindIII).All three fragments were gel purified, mixed and Prep-a-Gene purifiedprior to ligation. Following ligation and transformation, putativerecombinants were analyzed by restriction analysis; the EcoRI site wasfound to be regenerated at the fusion junction, as was predicted for afusion between the filled EcoRI and NcoI sites.

[0560] A construct encoding a fusion protein between the botulinal Cfragment gene and the MBP gene was constructed (i.e., this fusion lacksany C. difficile toxin A gene sequences) and termed pMBot. ThepMBot-construct was made by removal of the C. difficile toxin Asequences from the pMABot construct and fusing the C fragment genesequences to the MBP. This was accomplished by digestion of pMABot DNAwith StuI (located in the pMALc polylinker 5′ to the XbaI site) and XbaI(located 3′ to the NotI site at the toxA-Bot fusion junction), fillingin the XbaI site using the Klenow fragment, gel purifying the desiredrestriction fragment, and ligating the blunt ends to circularize theplasmid. Following ligation and transformation, putative recombinantswere analyzed by restriction mapping of the Bot insert (i.e, the C.botulinum C fragment sequences).

[0561] b) Expression of C. botulinum C Fragment Fusion Proteins in E.coli

[0562] Large scale (1 liter) cultures of the pMAL-c vector, and eachrecombinant construct described above in (a) were grown, induced, andsoluble protein fractions were isolated as described in Example 18. Thesoluble protein extracts were chromatographed on amylose affinitycolumns to isolate recombinant fusion protein. The purified recombinantfusion proteins were analyzed by running samples on SDS-PAGE gelsfollowed by Coomassie staining and by Western blot analysis as described[Williams et al, (1994) supra]. In brief, extracts were prepared andchromatographed in column buffer (10 mM NaPO₄, 0.5 M NaCl, 10 mMβ-mercaptoethanol, pH 7.2) over an amylose resin (New England Biolabs)column, and eluted with column buffer containing 10 mM maltose asdescribed [Williams, et al. (1994), supra]. An SDS-PAGE gel containingthe purified protein samples stained with Coomassie blue is shown inFIG. 26.

[0563] In FIG. 26, the following samples were loaded. Lanes 1-6 containprotein purified from E. coli containing the pMAL-c, pPA1870-2680,pMABot, pMNABot, pMCABot and pMBot plasmids, respectively. Lane 7contains broad range molecular weight protein markers (BioRad).

[0564] The protein samples were prepared for electrophoresis by mixing 5μl of eluted protein with 5 μl of 2× SDS-PAGE sample buffer (0.125 mMTris-HCl, pH 6.8, 2 mM EDTA, 6% SDS, 20% glycerol, 0.025% bromophenolblue; P-mercaptoethanol is added to 5% before use). The samples wereheated to 95° C. for 5 min, then cooled and loaded on a 7.5% agaroseSDS-PAGE gel. Broad range molecular weight protein markers were alsoloaded to allow estimation of the MW of identified fusion proteins.After electrophoresis, protein was detected generally by staining thegel with Coomassie blue.

[0565] In all cases the yields were in excess of 20 mg fusion proteinper liter culture (see Table 36) and, with the exception of the pMCABotprotein, a high percentage (i.e., greater than 20-50% of total elutedprotein) of the eluted fusion protein was of a MW predicted for the fulllength fusion protein (FIG. 26). It was estimated (by visual inspection)that less than 10% of the pMCABot fusion protein was expressed as thefull length fusion protein. TABLE 36 Yield Of Affinity Purified C.botulinum C Fragment/ MBP Fusion Proteins Yield (mg/liter Percentage OfTotal Construct of Culture) Soluble Protein pMABot 24 5.0 pMCABot 34 5.0pMNABot 40 5.5 pMBot 22 5.0 pMA1870-2680 40 4.8

[0566] These results demonstrate that high level expression of intact C.botulinum C fragment/C. difficile toxin A fusion proteins in E. coli isfeasible using the pMAL-c expression system. These results are incontrast to those reported by H. F. LaPenotiere, et al. (1993), supra.In addition, these results show that it is not necessary to fuse thebotulinal C fragment gene to the C. difficile toxin A gene in order toproduce a soluble fusion protein using the pMAL-c system in E. coli.

[0567] In order to determine whether the above-described botulinalfusion proteins were recognized by anti-C. botulinum toxin A antibodies,Western blots were performed. Samples containing affinity-purifiedproteins from E. coli containing the pMABot, pMCABot, pMNABot, pMBot,pMA1870-2680 or pMALc plasmids were analyzed. SDS-PAGE gels (7.5%acrylamide) were loaded with protein samples purified from eachexpression construct. After electrophoresis, the gels were blotted andprotein transfer was confirmed by Ponceau S staining (as described inExample 12b).

[0568] Following protein transfer, the blots were blocked by incubationfor 1 hr at 20° C. in blocking buffer [PBST (PBS containing 0.1% Tween20 and 5% dry milk)]. The blots were then incubated in 10 ml of asolution containing the primary antibody; this solution comprised a{fraction (1/500)} dilution of an anti-C. botulinum toxin A IgY PEG prep(described in Example 3) in blocking buffer. The blots were incubatedfor 1 hr at room temperature in the presence of the primary antibody.The blots were washed and developed using a rabbit anti-chicken alkalinephosphatase conjugate (Boehringer Mannheim) as the secondary antibody asfollows. The rabbit anti-chicken antibody was diluted to 1 μg/ml inblocking buffer (10 ml final volume per blot) and the blots wereincubated at room temperature for 1 hour in the presence of thesecondary antibody. The blots were then washed successively with PBST,BBS-Tween and 50 mM Na₂CO₃, pH 9.5. The blots were then developed infreshly-prepared alkaline phosphatase substrate buffer (100 μg/ml nitroblue tetrazolium, 50 μg/ml 5-bromo-chloro-indolylphosphate, 5 mM MgCl₂in 50 mM Na₂CO₃, pH 9.5). Development was stopped by flooding the blotswith distilled water and the blots were air dried.

[0569] This Western blot analysis detected anti-C. botulinum toxinreactive proteins in the pMABot, pMCABot, pMNABot and pMBot proteinsamples (corresponding to the predicted full length proteins identifiedabove by Coomassie staining in FIG. 26), but not in the pMA1100-2680 orpMALc protein samples.

[0570] These results demonstrate that the relevant fusion proteinspurified on an amylose resin as described above in section a) containedimmunoreactive C. botulinum C fragment protein as predicted.

EXAMPLE 23 Generation of Neutralizing Antibodies by Nasal Administrationof pMBot Protein

[0571] The ability of the recombinant botulinal toxin proteins producedin Example 22 to stimulate a systemic immune response against botulinaltoxin epitopes was assessed. This example involved: a) the evaluation ofthe induction of serum IgG titers produced by nasal or oraladministration of botulinal toxin-containing C. difficile toxin A fusionproteins and b) the in vivo neutralization of C. botulinum type Aneurotoxin by anti-recombinant C. botulinum C fragment antibodies.

[0572] a) Evaluation of the Induction of Serum IgG Titers Produced byNasal or Oral Administration of Botulinal Toxin-Containing C. difficileToxin A Fusion Proteins

[0573] Six groups containing five 6 week old CF female rats (CharlesRiver) per group were immunized nasally or orally with one of thefollowing three combinations using protein prepared in Example 22: (1)250 μg pMBot protein per rat (nasal and oral); 2) 250 μg pMABot proteinper rat (nasal and oral); 3) 125 μg pMBot admixed with 125 μgpMA1870-2680 per rat (nasal and oral). A second set of 5 groupscontaining 3 CF female rats/group were immunized nasally or orally withone of the following combinations (4) 250 μg pMNABot protein per rat(nasal and oral) or 5) 250 μg pMAL-c protein per rat (nasal and oral).

[0574] The fusion proteins were prepared for immunization as follows.The proteins (in column buffer containing 10 mM maltose) were diluted in0.1 M carbonate buffer, pH 9.5 and administered orally or nasally in a200 μl volume. The rats were lightly sedated with ether prior toadministration. The oral dosing was accomplished using a 20 gaugefeeding needle. The nasal dosing was performed using a P-200micro-pipettor (Gilson). The rats were boosted 14 days after the primaryimmunization using the techniques described above and were bled 7 dayslater. Rats from each group were lightly etherized and bled from thetail. The blood was allowed to clot at 37° C. for 1 hr and the serum wascollected.

[0575] The serum from individual rats was analyzed using an ELISA todetermine the anti-C. botulinum type A toxin IgG serum titer. The ELISAprotocol used is a modification of that described in Example 13c.Briefly, 96-well microtiter plates (Falcon, Pro-Bind Assay Plates) werecoated with C. botulinum type A toxoid (prepared as described in Example3a) by placing 100 μl volumes of C. botulinum type A toxoid at 2.5 μg/mlin PBS containing 0.005% thimerosal in each well and incubatingovernight at 4° C. The next morning, the coating suspensions weredecanted and all wells were washed three times using PBS.

[0576] In order to block non-specific binding sites, 100 μl of blockingsolution [0.5% BSA in PBS] was then added to each well and the plateswere incubated for 1 hr at 37° C. The blocking solution was decanted andduplicate samples of 150 μl of diluted rat serum added to the first wellof a dilution series. The initial testing serum dilution was 1:30 inblocking solution containing 0.5% Tween 20 followed by 5-fold dilutionsinto this solution. This was accomplished by serially transferring 30 μlaliquots to 120 μl blocking solution containing 0.5% Tween 20, mixing,and repeating the dilution into a fresh well. After the final dilution,30 μl was removed from the well such that all wells contained 120 μlfinal volume. A total of 3 such dilutions were performed (4 wellstotal). The plates were incubated 1 hr at 37° C. Following thisincubation, the serially diluted samples were decanted and the wellswere washed six times using PBS containing 0.5% Tween 20 (PBST). To eachwell, 100 μl of a rabbit anti-Rat IgG alkaline phosphatase (Sigma)diluted ({fraction (1/1000)}) in blocking buffer containing 0.5% Tween20 was added and the plate was incubated for 1 hr at 37° C. Theconjugate solutions were decanted and the plates were washed asdescribed above, substituting 50 mM Na₂CO₃, pH 9.5 for the PBST in thefinal wash. The plates were developed by the addition of −100 μl of asolution containing 1 mg/ml para-nitro phenyl phosphate (Sigma)dissolved in 50 mM Na₂CO₃, 10 mM MgCl₂, pH 9.5 to each well, andincubating the plates at room temperature in the dark for 5-45 min. Theabsorbency of each well was measured at 410 mm using a Dynatech MR 700plate reader. The results are summarized in Tables 37 and 38 andrepresent mean serum reactivities of individual mice. TABLE 37Determination Of Anti-C. botulinum Type A Toxin Serum IgG TitersFollowing Immunization With C. botulinum C Fragment- Containing FusionProteins Nasal Oral pMBot & pMBot & Route of Immunization pMA1870-pMA1870- Immunogen PREIMMUNE pMBot 2680 pMABot pMBot 2680 pMABotDilution 1:30  0.080 1.040 1.030 0.060 0.190 0.080 0.120 1:150  0.0170.580 0.540 0.022 0.070 0.020 0.027 1:750  0.009 0.280 0.260 0.010 0.0200.010 0.014 1:3750 0.007 0.084 0.090 0.009 0.009 0.010 0.007 # Rats 5 55 5 2 2 Tested

[0577] TABLE 38 Determination Of Anti-C. botulinum Type A Toxin SerumIgG Titers Following Immunization With C. botulinum CFragment-Containing Fusion Proteins Oral Route of Immunization NasalpMNA Immunogen PREIMMUNE pMBot pMABot pMNABot Bot Dilution 1:30 0.0400.557 0.010 0.015 0.010 1:150 0.009 0.383 0.001 0.003 0.002 1:750 0.0010.140 0.000 0.000 0.000 1:3750 0.000 0.040 0.000 0000 0.000 # Rats 1 1 33 Tested

[0578] The above ELISA results demonstrate that reactivity against thebotulinal fusion proteins was strongest when the route of administrationwas nasal; only weak responses were stimulated when the botulinal fusionproteins were given orally. Nasally delivered pMbot and pMBot admixedwith pMA1870-2680 invoked the greatest serum IgG response. These resultsshow that only the pMBot protein is necessary to induce this response,since the addition of the pMA1870-2680 protein did not enhance antibodyresponse (Table 37). Placement of the C. difficile toxin A fragmentbetween the MBP and the C. botulinum C fragment protein dramaticallyreduced anti-bot IgG titer (see results using pMABot, pMCABot andpMNABot proteins).

[0579] This study demonstrates that the pMBot protein induces a strongserum IgG response directed against C. botulinum type A toxin whennasally administered.

[0580] b) In Vivo Neutralization of C. botulinum Type A Neurotoxin byAnti-Recombinant C. botulinum C Fragment Antibodies

[0581] The ability of the anti-C. botulinum type A toxin antibodiesgenerated by nasal administration of recombinant botulinal fusionproteins in rats (Example 22) to neutralize C. botulinum type A toxinwas tested in a mouse neutralization model. The mouse model is the artaccepted method for detection of botulinal toxins in body fluids and forthe evaluation of anti-botulinal antibodies [E. J. Schantz and D. A.Kautter, J. Assoc. Off. Anal. Chem. 61:96 (1990) and Investigational NewDrug (BB-IND-3703) application by the Surgeon General of the Departmentof the Army to the Federal Food and Drug Administration]. The anti-C.botulinum type A toxin antibodies were prepared as follows.

[0582] Rats from the group given pMBot protein by nasal administrationwere boosted a second time with 250 μg pMBot protein per rat and serumwas collected 7 days later. Serum from one rat from this group and froma preimmune rat was tested for anti-C. botulinum type A toxinneutralizing activity in the mouse neutralization model described below.

[0583] The LD₅₀ of a solution of purified C. botulinum type A toxincomplex, obtained from Dr. Eric Johnson (University of WisconsinMadison), was determined using the intraperitoneal (IP) method ofSchantz and Kautter [J. Assoc. Off. Anal. Chem. 61:96 (1978)] using18-22 gram female ICR mice and was found to be 3500 LD₅₀/ml. Thedetermination of the LD₅₀ was performed as follows. A Type A toxinstandard was prepared by dissolving purified type A toxin complex in 25mM sodium phosphate buffer, pH 6.8 to yield a stock toxin solution of3.15×10⁷ LD₅₀/mg. The OD₂₇₈ of the solution was determined and theconcentration was adjusted to 10-20 μg/ml. The toxin solution was thendiluted 1:100 in gel-phosphate (30 mM phosphate, pH 6.4; 0.2% gelatin).Further dilutions of the toxin solution were made as shown below inTable 39. Two mice were injected IP with 0.5 ml of each dilution shownand the mice were observed for symptoms of botulism for a period of 72hours. TABLE 39 Determination Of The LD₅₀ Of Purified C. botulinum TypeA Toxin Complex Dilution Number Dead At 72 hr 1:320  2/2 1:640  2/21:1280 2/2 1:2560 0/2 (sick after 72 hr) 1:5120 0/2 (no symptoms)

[0584] From the results shown in Table 39, the toxin titer was assumedto be between 2560 LD₅₀/ml and 5120 LD₅₀/ml (or about 3840 LD₅₀/ml).This value was rounded to 3500 LD₅₀/ml for the sake of calculation.

[0585] The amount of neutralizing antibodies present in the serum ofrats immunized nasally with pMBot protein was then determined. Serumfrom two rats boosted with pMBot protein as described above andpreimmune serum from one rat was tested as follows. The toxin standardwas diluted 1:100 in gel-phosphate to a final concentration of 350LD₅₀/ml. One milliliter of the diluted toxin standard was mixed with 25μl of serum from each of the three rats and 0.2 ml of gel-phosphate. Themixtures were incubated at room temperature for 30 min with occasionalmixing. Each of two mice were injected with IP with 0.5 ml of themixtures. The mice were observed for signs of botulism for 72 hr. Micereceiving serum from rats immunized with pMBot protein neutralized thischallenge dose. Mice receiving preimmune rat serum died in less than 24hr.

[0586] The amount of neutralizing anti-toxin antibodies present in theserum of rats immunized with pMBot protein was then quantitated. Serumantibody titrations were performed by mixing 0.1 ml of each of theantibody dilutions (see Table 40) with 0.1 ml of a 1:10 dilution ofstock toxin solution (3.5×10⁴ LD₅₀/ml) with 1.0 ml of gel-phosphate andinjecting 0.5 ml IP into 2 mice per dilution. The mice were thenobserved for signs of botulism for 3 days (72 hr). The results aretabulated in Table 39.

[0587] As shown in Table 40 pMBot serum neutralized C. botulinum type Atoxin complex when used at a dilution of 1:320 or less. A meanneutralizing value of 168 IU/ml was obtained for the pMBot serum (an IUis defined as 10,000 mouse LD₅₀). This value translates to a circulatingserum titer of about 3.7 IU/mg of serum protein. This neutralizing titeris comparable to the commercially available bottled concentrated(Connaught Laboratories, Ltd.) horse anti-C. botulinum antiserum. A 10ml vial of Connaught antiserum contains about 200 mg/ml of protein; eachml can neutralize 750 IU of C. botulinum type A toxin. Afteradministration of one vial to a human, the circulating serum titer ofthe Connaught preparation would be approximately 25 IU/ml assuming anaverage serum volume of 3 liters). Thus, the circulating anti-C.botulinum titer seen in rats nasally immunized with pMBot protein (168IU/ml) is 6.7 time higher than the necessary circulation titer ofanti-C. botulinum antibody needed to be protective in humans. TABLE 40Quantitation Of Neutralizing Antibodies In pMBot Sera pMBot^(a) DilutionRat 1 Rat 2 1:20  2/2 2/2 1:40  2/2 2/2 1:80  2/2 2/2 1:160  2/2 2/21:320   2/2^(b)  2/2^(b) 1:640  0/2 0/2 1:1280 0/2 0/2 1:2560 0/2 0/2

[0588] These results demonstrate that antibodies capable of neutralizingC. botulinum type A toxin are induced when recombinant C. botulinum Cfragment fusion protein produced in E. coli is used as an immunogen.

EXAMPLE 24 Production of Soluble C. botulinum C Fragment ProteinSubstantially Free of Endotoxin Contamination

[0589] Example 23 demonstrated that neutralizing antibodies aregenerated by immunization with the pMBot protein expressed in E coli.These results showed that the pMBot fusion protein is a good vaccinecandidate. However, immunogens suitable for use as vaccines should bepyrogen-free in addition to having the capability of inducingneutralizing antibodies. Expression clones and conditions thatfacilitate the production of C. botulinum C fragment protein forutililization as a vaccine were developed.

[0590] The example involved: (a) determination of pyrogen content of thepMBot protein; (b) generation of C. botulinum C fragment protein free ofthe MBP; (c) expression of C. botulinum C fragment protein using variousexpression vectors; and (d) purification of soluble C. botulinum Cfragment protein substantially free of significant endotoxincontamination.

[0591] a) Determination of the Pyrogen Content of the pMBot Protein

[0592] In order to use a recombinant antigen as a vaccine in humans orother animals, the antigen preparation must be shown to be free ofpyrogens. The most significant pyrogen present in preparations ofrecombinant proteins produced in gram-negative bacteria, such as E.coli, is endotoxin [F.C. Pearson, Pyrogens: endotoxins, LAL testing anddepyrogentaion, (1985) Marcel Dekker, New York, pp. 23-56]. To evaluatethe utility of the pMBot protein as a vaccine candidate, the endotoxincontent in MBP fusion proteins was determined.

[0593] The endotoxin content of recombinant protein samples was assayedutilizing the Limulus assay (LAL kit; Associates of Cape Cod) accordingto the manufacturer's instructions. Samples of affinity-purified pMa1-cprotein and pMA1870-2680 were found to contain high levels of endotoxin[>50,000 EU/mg protein; EU (endotoxin unit)]. This suggested that MBP-or toxin A repeat-containing fusions with the botulinal C fragmentshould also contain high levels of endotoxin. Accordingly, removal ofendotoxin from affinity-purified pMa1-c and pMBot protein preparationswas attempted as follows.

[0594] Samples of pMa1-c and pMBot protein were depyrogenated withpolymyxin to determine if the endotoxin could be easily removed. Thefollowing amount of protein was treated: 29 ml at 4.8 OD₂₈₀/ml forpMa1-c and 19 mls at 1.44 OD₂₈₀ ml for pMBot. The protein samples weredialyzed extensively against PBS and mixed in a 50 ml tube (Falcon) with0.5 ml PBS-equilibrated polymyxin B (Affi-Prep Polymyxin, BioRad). Thesamples were allowed to mix by rotating the tubes overnight at 4° C. Thepolymyxin was pelleted by centrifugation for 30 min in a bench topcentrifuge at maximum speed (approximately 2000×g) and the supernatantwas removed. The recovered protein (in the supernatant) was quantifiedby OD₂₈₀, and the endotoxin activity was assayed by LAL. In both casesonly approximately ⅓ of the input protein was recovered and thepolymyxin-treated protein retained significant endotoxin contamination(approximately 7000 EU/mg of pMBot).

[0595] The depyrogenation experiment was repeated using an independentlypurified pMa1-c protein preparation and similar results were obtained.From these studies it was concluded that significant levels of endotoxincopurifies with these MBP fusion proteins using the amylose resin.Furthermore, this endotoxin cannot be easily removed by polymyxintreatment.

[0596] These results suggest that the presence of the MBP sequences onthe fusion protein complicated the removal of endotoxin frompreparations of the pMBot protein.

[0597] b) Generation of C. botulinum C Fragment Protein Free of the MBP

[0598] It was demonstrated that the pMBot fusion protein could not beeasily purified from contaminating endotoxin in section a) above. Theability to produce a pyrogen-free (e.g., endotoxin-free) preparation ofsoluble botulinal C fragment protein free of the MBP tag was nextinvestigated. The pMBot expression construct was designed to facilitatepurification of the botulinal C fragment from the MBP tag by cleavage ofthe fusion protein by utilizing an engineered Factor Xa cleavage sitepresent between the MBP and the botulinal C fragment. The Factor Xacleavage was performed as follows.

[0599] Factor Xa (New England Biolabs) was added to the pMBot protein(using a 0.1-1.0% Factor Xa/pMBot protein ratio) in a variety of bufferconditions [e.g., PBS-NaCl (PBS containing 0.5 M NaCl), PBS-NaClcontaining 0.2% Tween 20, PBS, PBS containing 0.2% Tween 20, PBS-C (PBScontaining 2 mM CaCl₂), PBS-C containing either 0.1 or 0.5% Tween 20,PBS-C containing either 0.1 or 0.5% NP-40, PBS-C containing either 0.1or 0.5% Triton X-100, PBS-C containing 0.1% sodium deoxycholate, PBS-Ccontaining 0.1% SDS]. The Factor Xa digestions were incubated for 12-72hrs at room temperature.

[0600] The extent of cleavage was assessed by Western blot or Coomassieblue staining of proteins following electrophoresis on denaturingSDS-PAGE gels, as described in Example 22. Cleavage reactions (andcontrol samples of uncleaved pMBot protein) were centrifuged for 2 minin a microfuge to remove insoluble protein prior to loading the sampleson the gel. The Factor Xa treated samples were compared with uncleaved,uncentrifuged pMBot samples on the same gel. The results of thisanalysis is summarized below.

[0601] 1) Most (about 90%) pMBot protein could be removed bycentrifugation, even when uncleaved control samples were utilized. Thisindicated that the pMBot fusion protein was not fully soluble (i.e., itexists as a suspension rather than as a solution). [This result wasconsistent with the observation that most affinity-purified pMBotprotein precipitates after long term storage (>2 weeks) at 4° C.Additionally, the majority (i.e., 75%) of induced pMBot protein remainsin the pellet after sonication and clarification of the induced E. coli.Resuspension of these insoluble pellets in PBS followed by sonicationresults in partial solubilization of the insoluble pMBot protein in thepellets.]

[0602] 2) The portion of pMBot protein that is fully in solution (about10% of pMBot protein) is completely cleaved by Factor Xa, but thecleaved (released) botulinal C fragment is relatively insoluble suchthat only the cleaved MBP remains fully in solution.

[0603] 3) None of the above reaction conditions enhanced solubilitywithout also reducing-effective cleavage. Conditions that effectivelysolubilized the cleaved botulinal C fragment were not identified.

[0604] 4) The use of 0.1% SDS in the buffer used for Factor Xa cleavageenhanced the solubility of the pMBot protein (all of pMBot protein wassoluble). However, the presence of the SDS prevented any cleavage of thefusion protein with Factor Xa.

[0605] 5) Analysis of pelleted protein from the cleavage reactionsindicated that both full length pMBot (i.e., uncleaved) and cleavedbotulinal C fragment protein precipitated during incubation.

[0606] These results demonstrate that purification of soluble botulinalC fragment protein after cleavage of the pMBot fusion protein iscomplicated by the insolubility of both the pMBot protein and thecleaved botulinal C fragment protein.

[0607] c) Expression of C. botulinum C Fragment Using Various ExpressionVectors

[0608] In order to determine if the solubility of the botulinal Cfragment was enhanced by expressing the C fragment protein as a nativeprotein, an N-terminal His-tagged protein or as a fusion withglutathione-S-transferase (GST), alternative expression plasmids wereconstructed. These expression constructs were generated utilizing themethodologies described in Example 22. FIG. 27 provides a schematicrepresentation of the vectors described below.

[0609] In FIG. 27, the following abbreviations are used. pP refers tothe pET23 vector. pHIS refers to the pETHisa vector. pBlue refers to thepBluescript vector. pM refers to the pMAL-c vector and pG refers to thepGEX3T vector (described in Example 11). The solid black lines representC. botulinum C fragment gene sequences; the solid black ovals representthe MBP; the hatched ovals represent GST; “HHHHH” represents thepoly-histidine tag. In FIG. 27, when the name for a restriction enzymeappears inside parenthesis, this indicates that the restriction site wasdestroyed during construction. An asterisk appearing with the name for arestriction enzyme indicates that this restriction site was recreated ata cloning junction.

[0610] i) Construction of pPBot

[0611] In order to express the C. botulinum C fragment as a native(i.e., non-fused) protein, the pPBot plasmid (shown schematically inFIG. 27) was constructed as follows. The C fragment sequences present inpAlterBot (Example 22) were removed by digestion of pAlterBot with NcoIand HindIII. The NcoI/HindIII C fragment insert was ligated to pETHisavector (described in Example 18b) which was digested with NcoI andHindIII. This ligation creates an expression construct in which theNcoI-encoded methionine of the botulinal C fragment is the initiatorcodon and directs expression of the native botulinal C fragment. Theligation products were used to transform competent BL21(DE3)pLysS cells(Novagen). Recombinant clones were identified by restriction mapping.

[0612] ii) Construction of pHisBot

[0613] In order to express the C. botulinum C fragment containing apoly-histidine tag at the amino-terminus of the recombinant protein, thepHisBot plasmid (shown schematically in FIG. 27) was constructed asfollows. The NcoI/HindIII botulinal C fragment insert from pAlterbot wasligated into the pETHisa vector which was digested with NheI andHindIII. The NcoI (on the C fragment insert) and NheI (on the pETHisavector) sites were filled in using the Klenow fragment prior toligation; these sites were then blunt end ligated (the NdeI site wasregenerated at the clone junction as predicted). The ligation productswere used to transform competent BL21(DE3)pLysS cells and recombinantclones were identified by restriction mapping.

[0614] The resulting pHisBot clone expresses the botulinal C fragmentprotein with a histidine-tagged N-terminal extension having thefollowing sequence: MetGlyH is HisHisHisHisHisHisHisHisHisSerSerGlyHisIleGluGlyArgHisMetAla (SEQ IDNO:24); the amino acids encoded by the botulinal C fragment gene areunderlined and the vector encoded amino acids are presented in plaintype. The nucleotide sequence present in the pETHisa vector whichencodes the pHisBot fusion protein is listed in —SEQ ID NO:25. The aminoacid sequence of the pHisBot protein is listed in SEQ ID NO:26.

[0615] iii) Construction of pGBot

[0616] The botulinal C fragment protein was expressed as a fusion withthe glutathione-S-transferase protein by constructing the pGBot plasmid(shown schematically in FIG. 27). This expression construct was createdby cloning the NotI/SalI C fragment insert present in pBlueBot (Example22) into the pGEX3T vector which was digested with SmaI and XhoI. TheNotI site (present on the botulinal fragment) was made blunt prior toligation using the Klenow fragment. The ligation products were used totransform competent BL21 cells.

[0617] Each of the above expression constructs were tested byrestriction digestion to confirm the integrity of the constructs.

[0618] Large scale (1 liter) cultures of pPBot [BL21(DE3)pLysS host],pHisBot [BL21(DE3)pLysS host] and pGBot (BL21 host) were grown in 2× YTmedium and induced (using IPTG to 0.8-1.0 mM) for 3 hrs as described inExample 22. Total, soluble and insoluble protein preparations wereprepared from 1 ml aliquots of each large scale culture [Williams et al.(1994), supra] and analyzed by SDS-PAGE. No obvious induced band wasdetectable in the pPBot or pHisBot samples by Coomassie staining, whilea prominent insoluble band of the anticipated MW was detected in thepGBot sample. Soluble lysates of the pGBot large scale (resuspended inPBS) or pHisBot large scale [resuspended in Novagen 1× binding buffer (5mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9)] cultures wereprepared and used to affinity purify soluble affinity-tagged protein asfollows.

[0619] The pGBot lysate was affinity purified on a glutathione-agaroseresin (Pharmacia) exactly as described in Smith and Corcoran [CurrentProtocols in Molecular Biology, Supplement 28 (1994), pp.16.7.1-16.7.7]. The pHisBot protein was purified on the His-Bind resin(Novagen) utilizing the His-bind buffer kit (Novagen) exactly asdescribed by manufacturer.

[0620] Samples from the purification of both the pGBot and pHisBotproteins (including uninduced, induced, total, soluble, andaffinity-purified eluted protein) were resolved on SDS-PAGE gels.Following electrophoresis, proteins were analyzed by Coomassie stainingor by Western blot detection utilizing a chicken anti-C. botulinum TypeA toxoid antibody (as described in Example 22).

[0621] These studies showed that the pGBot protein was almost entirelyinsoluble under the utilized conditions, while the pHisBot protein wassoluble. Affinity purification of the pHisBot protein on this firstattempt was inefficient, both in terms of yield (most of theimmunoreactive botulinal protein did not bind to the His-bind resin) andpurity (the botulinal protein was estimated to comprise approximately20% of the total eluted protein).

[0622] d) Purification of Soluble C. botulinum C Fragment ProteinSubstantially Free of Endotoxin Contamination

[0623] The above studies showed that the pHisBot protein was expressedin E. coli as a soluble protein. However, the affinity purification ofthis protein on the His-bind resin was very inefficient. In order toimprove the affinity purification of the soluble pHisBot protein (interms of both yield and purity), an alternative poly-histidine bindingaffinity resin (Ni-NTA resin; Qiagen) was utilized. The Ni-NTA resin wasreported to have a superior binding affinity (Kd=1×10⁻¹³ at pH 8.0;Qiagen user manual) relative to the His-bind resin.

[0624] A soluble lysate (in Novagen IX binding buffer) from an induced 1liter 2× YT culture was prepared as described above. Briefly, theculture of pHisBot [Bl21(DE3)pLysS host] was grown at 37° C. to an OD₆₀₀of 0.7 in 1 liter of 2× YT medium containing 100 μg/ml ampicillin, 34μg/ml chloramphenicol and 0.2% glucose. Protein expression was inducedby the addition of IPTG to 1 mM. Three hours after the addition of theIPTG, the cells were cooled for 15 min in a ice water bath and thencentrifuged 10 min at 5000 rpm in a JA10 rotor (Beckman) at 4° C. Thepellets were resuspended in a total volume of 40 mls Novagen 1× bindingbuffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9), transferredto two 35 ml Oakridge tubes and frozen at −70° C. for at least 1 hr. Thetubes were thawed and the cells were lysed by sonication (4×20 secondbursts using a Branson Sonifier 450 with a power setting of 6-7) on ice.The suspension was clarified by centrifugation for 20 min at 9,000 rpm(10,000×g) in a JA-17 rotor (Beckman).

[0625] The soluble lysate was brought to 0.1% NP40 and then was batchabsorbed to 7 ml of a 1:1 slurry of Ni-NTA resin:binding buffer bystirring for 1 hr at 4° C. The slurry was poured into a column having aninternal diameter of 1 or 2.5 cm (BioRad). The column was then washedsequentially with 15 mls of Novagen IX binding buffer containing 0.1%NP40, 15 ml of Novagen 1× binding buffer, 15 ml wash buffer (60 mMimidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) and 15 ml NaHPO₄ washbuffer (50 mM NaHPO₄, pH 7.0, 0.3 M NaCl, 10% glycerol). The boundprotein was eluted by protonation of the resin using elution buffer (50mM NaHPO₄, pH 4.0, 0.3 M NaCl, 10% glycerol). The eluted protein wasstored at 4° C.

[0626] Samples of total, soluble and eluted protein were resolved bySDS-PAGE. Protein samples were prepared for electrophoresis as describedin Example 22b. Duplicate gels were stained with Coomassie blue tovisualize the resolved proteins and C. botulinum type A toxin-reactiveprotein was detected by Western blot analysis as described in Example22b. A representative Coomassie stained gel is shown in FIG. 28. In FIG.28, the following samples were loaded on the 12.5% acrylamide gel. Lanes1-4 contain respectively total protein, soluble protein, soluble proteinpresent in the flow-through of the Ni-NTA column and affinity-purifiedpHisBot protein (i.e., protein released from the Ni-NTA resin byprotonation). Lane 5 contains high molecular weight protein markers(BioRad).

[0627] The purification of pHisBot protein resulted in a yield of 7 mgof affinity purified protein from a 1 liter starting culture ofBL21(DE3)pLysS cells harboring the pHisBot plasmid. The yield ofpurified pHisBot protein represented approximately 0.4% of the totalsoluble protein in the induced culture. Analysis of the purified pHisBotprotein by SDS-PAGE revealed that at least 90-95% of the protein waspresent as a single band (FIG. 28) of the predicted MW (50 kD). This 50kD protein band was immunoreactive with anti-C. botulinum type A toxinantibodies. The extinction coefficient of the protein preparation wasdetermined to be 1.4 (using the Pierce BCA assay) or 1.45 (using theLowry assay) OD₂₈₀ per 1 mg/ml solution.

[0628] Samples of pH neutralized eluted pHisBot protein were resolved ona KB 803 HPLC column (Shodex). Although His-tagged proteins are retainedby this sizing column (perhaps due to the inherent metal binding abilityof the proteins), the relative mobility of the pHisBot protein wasconsistent with that expected for a non-aggregated protein in solution.Most of the induced pHisBot protein was determined to be soluble underthe growth and solubilization conditions utilized above (i.e., greaterthan 90% of the pHisBot protein was found to be soluble as judged bycomparison of the levels of pHisBot protein seen in total and solubleprotein samples prepared from BL21(DE3)pLysS cells containing thepHisBot plasmid). SDS-PAGE analysis of samples obtained aftercentrifugation, extended storage at −20° C., and at least 2 cycles offreezing and thawing detected no protein loss (due to precipitation),indicating that the pHisBot protein is soluble in the elution buffer(i.e., 50 mM NaHPO₄, pH 4.0, 0.3 M NaCl, 10% glycerol).

[0629] Determination of endotoxin contamination in the affinity purifiedpHisBot preparation (after pH neutralization) using the LAL assay(Associates of Cape Cod) detected no significant endotoxincontamination. The assay was performed using the endpoint chromogenicmethod (without diazo-coupling) according to the manufacturer'sinstructions. This method can detect concentrations of endotoxin greaterthan or equal to 0.03 EU/ml (EU refers to endotoxin units). The LALassay was run using 0.5 ml of a solution comprising 0.5 mg pHisBotprotein in 50 mM NaHPO₄, pH 7.0, 0.3 M NaCl, 10% glycerol; 30-60 EU weredetected in the 0.5 ml sample. Therefore, the affinity purified pHisBotpreparation contains 60-120 EU/mg of protein. FDA Guidelines for theadministration of parenteral drugs require that a composition to beadministered to a human contain less than 5 EU/kg body weight (Theaverage human body weight is 70 kg; therefore up to 349 EU units can bedelivered in a parental dose.). Because very small amount of protein areadministered in a vaccine preparation (generally in the range of 10-500μg of protein), administration of affinity purified pHisBot containing60-120 EU/mg protein would result in delivery of only a small percentageof the permissible endotoxin load. For example, administration of 10-500μg of purified pHisBot to a 70 kg human, where the protein preparationcontains 60 EU/mg protein, results in the introduction of only 0.6 to 30EU [i.e., 0.2 to 8.6% of the maximum allowable endotoxin burden perparenteral dose (less than 5 EU/kg body weight)].

[0630] The above results demonstrate that endotoxin (LPS) does notcopurify with the pHisBot protein using the above purification scheme.Preparations of recombinantly produced pHisBot protein containing lowerlevels of endotoxin (less than or equal to 2 EU/mg recombinant protein)may be produced by washing the Ni-NTA column with wash buffer until theOD₂₈₀ returns to baseline levels (i.e., until no more UV-absorbingmaterial comes off of the column).

[0631] The above results illustrate a method for the production andpurification of soluble, botulinal C fragment protein substantially freeof endotoxin.

EXAMPLE 25 Optimization of the Expression and Purification of pHisBotProtein

[0632] The results shown in Example 24d demonstrated that the pHisBotprotein is an excellent candidate for use as a vaccine as it could beproduced as a soluble protein in E. coli and could be purified free ofpyrogen activity. In order to optimize the, expression and purificationof the pHisBot protein, a variety of growth and purification conditionswere tested.

[0633] a) Growth Parameters

[0634] i) Host Strains

[0635] The influence of the host strain utilized upon the production ofsoluble pHisBot protein was investigated. A large scale purification ofpHisBot was performed [as described in Example 24d above] using theBL21(DE3) host (Novagen) rather than the BL21(DE3)pLysS host. Thedeletion of the pLysS plasmid in the BL21(DE3) host yielded higherlevels of expression due to de-repression of the plasmid's T7-lacpromoter. However, the yield of affinity-purified soluble recombinantprotein was very low (approximately 600 μg/liter culture) when purifiedunder conditions identical to those described in Example 24d above. Thisresult was due to the fact that expression in the BL21(DE3) host yieldedvery high level expression of the pHisBot protein as insoluble inclusionbodies as shown by SDS-PAGE analysis of protein prepared from inducedBL21(DE3) cultures (FIG. 29, lanes 1-7, described below). These resultsdemonstrate that the pHisBot protein is not inherently toxic to E. colicells and can be expressed to high levels using the appropriatepromoter/host combination.

[0636]FIG. 29 shows a Coomassie blue stained SDS-PAGE gel (12.5%acrylamide) onto which extracts prepared from BL21(DE3) cells containingthe pHisBot plasmid were loaded. Each lane was loaded with 2.5 μlprotein sample mixed with 2.5 μl of 2× SDS sample buffer. The sampleswere handled as described in Example 22b. The following samples wereapplied to the gel. Lanes 1-7 contain protein isolated from theBL21(DE3) host. Lanes 8-14 contain proteins isolated from theBL21(DE3)pLysS host. Total protein was loaded in lanes 1, 2, 4, 6, 8, 10and 12. Soluble protein was loaded in Lanes 3, 5, 7, 9, 11 and 13. Lane1 contains protein from uninduced host cells. Lanes 2-13 contain proteinfrom host cells induced for 3 hours. IPTG was added to a finalconcentration of 0.1 mM (Lanes 6-7), 0.3 mM (Lanes 4-5) or 1.0 mM (Lanes2, 3, 8-13). The cultures were grown in LB broth (Lanes 8-9), 2× YTbroth (Lanes 10-11) or terrific broth (Lanes 1-7, 12-13). The pHisBotprotein seen in Lanes 3, 5 and 7 is insoluble protein which spilled overfrom Lanes 2, 4 and 6, respectively. High molecular weight proteinmarkers (BioRad) were loaded in Lane 14.

[0637] A variety of expression conditions were tested to determine ifthe BL21(DE3) host could be utilized to express soluble pHisBot proteinat suitably high levels (i.e, about 10 mg/ml). The conditions alteredwere temperature (growth at 37 or 30° C.), culture medium (2× YT, LB orTerrific broth) and inducer levels (0.1, 0.3 or 1.0 mM IPTG). Allcombinations of these variables were tested and the induction levels andsolubility was then assessed by S9S-PAGE analysis of total and solubleextracts [prepared from 1 ml samples as described in Williams et al.,(1994), supra].

[0638] All cultures were grown in 15 ml tubes (Falcon #2057). Allculture medium was prewarmed overnight at the appropriate temperatureand were supplemented with 100 μg/ml ampicillin and 0.2% glucose.Terrific broth contains 12 g/l bacto-tryptone, 24 g/l bacto-yeastextract and 100 ml/l of a solution comprising 0.17 M KH₂PO₄, 0.72 MK₂HPO₄. Cultures were grown in a incubator on a rotating wheel (toensure aeration) to an OD₆₀₀ of approximately 0.4, and induced by theaddition of IPTG. In all cases, high level expression of insolublepHisBot protein was observed, regardless of temperature, medium orinducer concentration.

[0639] The effect of varying the concentration of IPTG upon 2× YTcultures grown at 23° C. was then investigated. IPTG was added to afinal concentration of either 1 mM, 0.1 mM, 0.05 μM or 0.01 nM. At thistemperature, similar levels of pHis Bot protein was induced in thepresence of either 1 or 0.1 mM IPTG; these levels of expression waslower than that observed at higher temperatures. Induced protein levelswere reduced at 0.05 mM IPTG and absent at 0.01 mM IPTG (relative to 1.0and 0.1 mM IPTG inductions at 23° C.). However, no conditions wereobserved in which the induced pHisBot protein was soluble in this host.Thus, although expression levels are superior in the BL21(DE3) host (ascompared to the BL21(DE3)pLysS host), conditions that facilitate theproduction of soluble protein in this host could not be identified.

[0640] These results demonstrate that production of soluble pHisBotprotein was achieved using the BL21(DE3)pLysS host in conjunction withthe T7-lac promoter.

[0641] ii) Effect of Varying Temperature, Medium and IPTG Concentrationand Length of Induction

[0642] The effect growing the host cells in various mediums upon theexpression of recombinant botulinal protein from the pHisBot expressionconstruct [in the BL21(DE3)pLysS host] was investigated. BL21(DE3)pLysScells containing the pHisBot plasmid were grown in either LB, 2× YT orTerrific broth at 37° C. The cells were induced using 1 mM IPTG for a 3hr induction period. Expression of pHisBot protein was found to be thehighest when the cells were grown in 2× YT broth (see FIG. 29, lanes8-13).

[0643] The cells were then grown at 30° C. in 2× YT broth and theconcentration of IPTG was varied from 1.0, 0.3 or 0.1 mM and the lengthof induction was either 3 or 5 hours. Expression of pHisBot protein wassimilar at all 3 inducer concentrations utilized and the levels ofinduced protein were higher after a 5 hr induction as compared to a 3 hrinduction.

[0644] Using the conditions found to be optimal for the expression ofpHisBot protein, a large scale culture was grown in order to providesufficient material for a large scale purification of the pHisBotprotein. Three 1 liter cultures were grown in 2× YT medium containing100 μg/ml ampicillin, 34 μg/ml chloramphenicol and 0.2% glucose. Thecultures were grown at 30° C. and were induced with 1.0 mM IPTG for a 5hr period. The cultures were harvested and a soluble lysate wereprepared as described in Example 18. A large scale purification wasperformed as described in Example 24d with the exception that except thesoluble lysate was batch absorbed for 3 hours rather than for 1 hour.The final yield was 13 mg pHisBot protein/liter culture. The pHisBotprotein represented 0.75% of the total soluble protein.

[0645] The above results demonstrate growth conditions under whichsoluble pHisBot protein is produced (i.e., use of the BL21(DE3)pLysShost, 2× YT medium, 30° C., 1.0 mM IPTG for 5 hours).

[0646] b) Optimization Of Purification Parameters

[0647] For optimization of purification conditions, large scale cultures(3×1 liter) were grown at 30° C. and induced with 1 mM IPTG for 5 hoursas described above. The cultures were pooled, distributed to centrifugebottles, cooled and pelleted as described in Example 24d. The cellpellets were frozen at −70° C. until used. Each cell pellet represented⅓ of a liter starting culture and individual bottles were utilized foreach optimization experiment described below. This standardized theinput bacteria used for each experiment, such that the yields ofaffinity purified pHisBot protein could be compared between differentoptimization experiments.

[0648] i) Binding Specificity (pH Protonation)

[0649] A lysate of pHisBot culture was prepared in PBS (pH 8.0) andapplied to a 3 ml Ni-NTA column equilibrated in PBS (pH 8.0) using aflow rate of 0.2 ml/min (3-4 column volumes/hr) using an Econochromatography system (BioRad). The column was washed with PBS (pH 8.0)until the absorbance (OD₂₈₀) of the elute was at baseline levels. Theflow rate was then increased to 2 ml/min and the column was equilibratedin PBS (pH 7.0). A pH gradient (pH 7.0 to 4.0 in PBS) was applied inorder to elute the bound pHisBot protein from the column. Fractions werecollected and aliquots were resolved on SDS-PAGE gels. The PAGE gelswere subjected to Western blotting and the pHisBot protein was detectedusing a chicken anti-C. botulinum Type A toxoid antibody as described inExample 22.

[0650] From the Western blot analysis it was determined that the pHisBotprotein begins to elute from the Ni-NTA column at pH 6.0. This isconsistent with the predicted elution of a His-tagged protein monomer atpH 5.9.

[0651] These results demonstrate that the pH at which the pHisBotprotein is protonated (released) from Ni-NTA resin in PBS buffer is pH6.0.

[0652] ii) Binding Specificity (Imidazole Competition)

[0653] In order to define purification conditions under which the nativeE. coli proteins could be removed from the Ni-NTA column while leavingthe pHisBot protein bound to the column, the following experiment wasperformed. A lysate of pHisBot culture was prepared in 50 mM NaHPO₄, 0.5M NaCl, 8 mM imidazole (pH 7.0). This lysate was applied to a 3 mlNi-NTA column equilibrated in 50 mM NaHPO₄, 0.5 M NaCl (pH 7.0) using anEcono chromatography system (BioRad). A flow rate of 0.2 ml/min (3-4column volumes/hr) was utilized. The column was washed with 50 mMNaHPO₄, 0.5 M NaCl (pH 7.0) until the absorbance of the elute returnedto baseline. The flow rate was then increased to 2 ml/min.

[0654] The column was eluted using an imidazole step gradient [in 50 mMNaHPO₄, 0.5 M NaCl (pH 7.0)]. Elution steps were 20 mM, 40 mM, 60 mM, 80mM, 100 mM, 200 mM, 1.0 M imidazole, followed by a wash using 0.1 mMEDTA (to strip the nickel from the column and remove any remainingprotein). In each step, the wash was continued until the OD₂₈₀ returnedto baseline. Fractions were resolved on SDS-PAGE gels, Western blotted,and pHisBot protein detected using a chicken anti-C. botulinum Type Atoxoid antibody as described in Example 22. Duplicate gels were stainedwith Coomassie blue to detect eluted protein in each fraction.

[0655] The results of the PAGE analysis showed that most of thenon-specifically binding bacterial protein was removed by the 20 mMimidiazole wash, with the remaining bacterial proteins being removed inthe 40 and 60 mM imidazole washes. The pHisBot protein began to elute at100 mM imidazole and was quantitatively eluted in 200 mM imidazole.

[0656] These results precisely defined the window of imidazole washstringency that optimally removes E. coli proteins from the column whilespecifically retaining the pHisBot protein in this buffer. These resultsprovided conditions under which the pHisBot protein can be purified freeof contaminating host proteins.

[0657] iii) Purification Buffers and Optimized Purification Protocols

[0658] A variety of purification parameters were tested during thedevelopment of an optimized protocol for batch purification of solublepHisBot protein. The results of these analyses are summarized below.

[0659] Batch purifications were performed (as described in Example 24d)using several buffers to determine if alternative buffers could beutilized for binding of the pHisBot protein to the Ni-NTA column. It wasdetermined that quantitative binding of pHisBot protein to the Ni-NTAresin was achieved in either Tris-HCl (pH 7.9) or NaHPO₄ (pH 8.0)buffers. Binding of the pHisBot protein in NaHPO₄ buffer was notinhibited using 5 mM, 8 mM or 60 mM imidazole. Quantitative elution ofbound pHisBot protein was obtained in buffers containing 50 mM NaHPO₄,0.3 M NaCl (pH 3.5-4.0), with or without 10% glycerol. However,quantitation of soluble affinity purified pHisBot protein before andafter a freeze thaw (following several weeks storage of the affinitypurified elute at −20° C.) revealed that 94% of the protein wasrecovered using the glycerol-containing buffer, but only 68% of theprotein was recovered when the buffer lacking glycerol was employed.This demonstrates that glycerol enhanced the solubility of the pHisBotprotein in this low pH buffer when the eluted protein was stored atfreezing temperatures (e.g., −20° C.). Neutralization of pH by additionof NaH₂PO₄ buffer did not result in obvious protein precipitation.

[0660] It was determined that quantitative binding of pHisBot proteinusing the batch format occurred after 3 hrs (FIG. 30), but not after 1hr of binding at 4° C. (the resin was stirred during binding). FIG. 30depicts a Coomaisse blue stained SDS-PAGE gel (7.5% acrylamide)containing samples of proteins isolated during the purification ofpHisBot protein from lysate prepared from the BL21(DE3)pLysS host. Eachlane was loaded with 5 μl of protein sample mixed with 5 μl of 2× samplebuffer and processed as described in Example 22b. Lane 1 contains highmolecular weight protein markers (BioRad). Lanes 2 and 3 contain proteineluted from the Ni-NTA resin. Lane 4 contains soluble protein after a 3hr batch incubation with the Ni-NTA resin. Lanes 5 and 6 contain solubleand total protein, respectively. FIG. 30 demonstrates that the pHisBotprotein is completely soluble [compare Lanes 5 and 6 which show that asimilar amount of the 50 kD pHisBot protein is seen in both; if asubstantial amount (greater than 20%) of the pHisBot protein werepartially insoluble in the host cell, more pHisBot protein would be seenin lane 6 (total protein) as compared to lane 5 (soluble protein)]. FIG.30 also demonstrates that the pHisBot protein is completely removed fromthe lysate after batch absorption with the Ni-NTA resin for 3 hours(compare Lanes 4 and 5).

[0661] The reported high affinity interaction of the Ni-NTA resin withHis-tagged proteins (K_(d)=1×10⁻¹³ at pH 8.0) suggested that it shouldbe possible to manipulate the resin-protein complexes withoutsignificant release of the bound protein. Indeed, it was determined thatafter the recombinant protein was bound to the Ni-NTA resin, theresin-pHisBot protein complex was highly stable and remained boundfollowing repeated rounds of centrifugation of the resin for 2 min at1600×g. When this centrifugation step was performed in a 50 ml tube(Falcon), a tight resin pellet formed. This allowed the removal of spentsoluble lysate by pouring off the supernatant followed by resuspensionof the pellet in wash buffer. Further washes can be performed bycentrifugation. The ability to perform additional washes permits thedevelopment of protocols for batch absorption of large volumes of lysatewith removal of the lysate being performed simply by centrifugationfollowing binding of the recombinant protein to the resin.

[0662] A simplified, integrated purification protocol was developed asfollows. A soluble lysate was made by resuspending the induced cellpellet in binding buffer [50 mM NaHPO₄, 0.5 M NaCl, 60 mM imidazole (pH8.0)], sonicating 4×20 sec and centrifuging for 20 min at 10,000×g.NP-40 was added to 0.1% and Ni-NTA resin (equilibrated in bindingbuffer) was added. Eight milliliters of a 1:1 slurry (resin:bindingbuffer) was used per liter of starting culture. The mixture was stirredfor 3 hrs at 4° C. The slurry was poured into a column having a 1 cminternal diameter (BioRad), washed with binding buffer containing 0.1%NP40, then binding buffer until baseline was established (these stepsmay alternatively be performed by centrifugation of the resin,resuspension in binding buffer containing NP40 followed bycentrifugation and resuspension in binding buffer). Imidazole wasremoved by washing the resin with 50 mM NaHPO₄, 0.3M NaCl (pH 7.0).Protein bound to the resin was eluted using the same buffer (50 mMNaHPO₄, 0.3M NaCl) having a reduced pH (pH 3.5-4.0).

[0663] A pilot purification was performed following this protocol andyielded 18 mg/liter affinity-purified pHisBot. The pHisBot protein wasgreater than 90% pure as estimated by Coomassie staining of an SDS-PAGEgel. This represents the highest observed yield of solubleaffinity-purified pHisBot protein and this protocol eliminates the needfor separate imidazole-containing binding and wash buffers. In additionto providing a simplified and efficient protocol for the affinitypurification of recombinant pHisBot protein, the above results provide avariety of purification conditions under which pHisBot protein can beisolated.

EXAMPLE 26 The pHisBot Protein is an Effective Immunogen

[0664] In Example 23 it was demonstrated that neutralizing antibodiesare generated in mouse serum after nasal immunization with the pMBotprotein. However, the pMBot protein was found to copurify withsignificant amounts of endotoxin which could not be easily removed. ThepHisBot protein, in contrast, could be isolated free of significantendotoxin contamination making pHisBot a superior candidate for vaccineproduction. To further assess the suitability of pHisBot as a vaccine,the immunogenicity of the pHisBot protein was determined and acomparison of the relative immunogenicity of pMBot and pHisBot proteinsin mice was performed as follows.

[0665] Two groups of eight BALBc mice were immunized with either pMBotprotein or pHisBot protein using Gerbu GMDP adjuvant (CC Biotech). pMBotprotein (in PBS containing 10 mM maltose) or pHisBot protein (in 50mMNaHPO₄, 0.3 M NaCl, 10% glycerol, pH 4.0) was mixed with Gerbuadjuvant and used to immunize mice. Each mouse received an IP injectionof 100 μl antigen/adjuvant mix (50 μg antigen plus 1 μg adjuvant) on day0. Mice were boosted as described above with the exception that theroute of administration was IM on day 14 and 28. The mice were bled onday 77 and anti-C. botulinum Type A toxoid titers were determined usingserum collected from individual mice in each group (as described inExample 23). The results are shown in Table 41. TABLE 41 Anti-C.botulinum Type A Toxoid Serum IgG Titers In Individual Mice ImmunizedWith pMBot or pHisBot Protein Preimmune¹ pMBot² pHisBot² Sample DilutionSample Dilution Sample Dilution Mouse # 1:50 1:250 1:1250 1:6250 1:501:250 1:1250 1:6250 1:50 1:250 1:1250 1:620 1 0.678 0.190 0.055 0.0071.574 0.799 0.320 0.093 2 1.161 0.931 0.254 0.075 1.513 0.829 0.4090.134 3 1.364 0.458 0.195 0.041 1.596 1.028 0.453 0.122 4 1.622 1.1890.334 0.067 1.552 0.840 0.348 0.090 5 1.612 1.030 0.289 0.067 1.6291.580 0.895 0.233 6 0.913 0.242 0.069 0.013 1.485 0.952 0.477 0.145 70.910 0.235 0.058 0.014 1.524 0.725 0.269 0.069 8 0.747 0.234 0.0580.014 1.274 0.427 0.116 0.029 Mean 0.048 0.021 0.011 0.002 1.133 0.5640.164 0.037 1.518 0.896 0.411 0.114 Titer

[0666] The results shown above in Table 41 demonstrate that both thepMBot and pHisBot proteins are immunogenic in mice as 100% of the mice(8/8) in each group seroconverted from non-immune to immune status. Theresults also show that the average titer of anti-C. botulinum Type Atoxoid IgG is 2-3 fold higher after immunization with the pHisBotprotein relative to immunization with the pMBot protein. This suggeststhat the pHisBot protein may be a superior immunogen to the pMBotprotein.

EXAMPLE 27 Immunization with the Recombinant pHisBot Protein GeneratesNeutralizing Antibodies

[0667] The results shown in Example 26 demonstrated that both thepHisBot and pMBot proteins were capable of inducing high titers ofanti-C. botulinum type A toxoid-reactive antibodies in immunized hosts.The ability of the immune sera from mice immunized with either thepHisBot or pMBot proteins to neutralize C. botulinum type A toxoid invivo was determined using the mouse neutralization assay described inExample 23b.

[0668] The two groups of eight BALBc mice immunized with either pMBotprotein or pHisBot protein in Example 26 were boosted again one weekafter the bleeding on day 77. The boost was performed by mixing pMBotprotein (in PBS containing 10 mM maltose) or pHisBot protein (in 50 mMNaHPO₄, 0.3 M NaCl, 10% glycerol, pH 4.0) with Gerbu adjuvant asdescribed in Example 26. Each mouse received an IP injection of 100 μlantigen/adjuvant mix (50 μg antigen plus 1 μg adjuvant). The mice werebled 6 days after this boost and the serum from mice within a group waspooled. Serum from preimmune mice was also collected (this serum is thesame serum described in the footnote to Table 41).

[0669] The presence of neutralizing antibodies in the pooled orpreimmune serum was detected by challenging mice with 5 LD₅₀ units oftype A toxin mixed with 100 μl of pooled serum. The challenge wasperformed by mixing (per mouse to be injected) 100 μl of serum from eachpool with 100 μl of purified type A toxin standard (50 LD₅₀/ml preparedas described in Example 23b) and 500 μl of gel-phosphate. The mixtureswere incubated for 30 min at room temperature with occasional mixing.Each of four mice were injected IP with the mixtures (0.7 ml/mouse). Themice were observed for signs of botulism for 72 hours. Mice receivingtoxin mixed with serum from mice immunized with either the pHisBot orpMBot proteins showed no signs of botulism intoxication. In contrast,mice receiving preimmune serum died in less than 24 hours.

[0670] These results demonstrate that antibodies capable of neutralizingC. botulinum type A toxin are induced when either of the recombinant C.botulinum C fragment proteins pHisBot or pMBot are used as immunogens.

EXAMPLE 28 Cloning and Expression of the C Fragment of C. botulinumSerotype A Toxin in E. coli Utilizing A Native Gene Fragment

[0671] In Example 22 above, a synthetic gene was used to express the Cfragment of C. botulinum serotype A toxin in E. coli. The synthetic genereplaced non-preferred (i.e., rare) codons present in the C fragmentgene with codons which are preferred by E. coli. The synthetic gene wasgenerated because it was been reported that genes which have a high A/Tcontent (such as most clostridial genes) creates expression difficultiesin E. coli and yeast. Furthermore, LaPenotiere et al. suggested thatproblems encountered with the stability (non-fusion constructs) andsolubility (MBP fusion constructs) of the C fragment of C. botulinumserotype A toxin when expressed in E. coli was most likely due to theextreme A/T richness of the native C. botulinum serotype A toxin genesequences (LaPenotiere, et al, supra).

[0672] In this example, it was demonstrated that successful expressionof the C fragment of C. botulinum type A toxin gene in E. coli does notrequire the elimination of rare codons (i.e., there is no need to use asynthetic gene). This example involved a) the cloning of the native Cfragment of the C. botulinum serotype A toxin gene and construction ofan expression vector and b) a comparison of the expression andpurification yields of C. botulinum serotype A C fragments derived fromnative and synthetic expression vectors.

[0673] a) Cloning of the Native C Fragment of the C. botulinum SerotypeA Toxin Gene and Construction of an Expression Vector

[0674] The serotype A toxin gene was cloned from C. botulinum genomicDNA using PCR amplification. The following primer pair was employed:5′-CGCCATGGCTAG ATTATTATCTACATTTAC-3′ (5′ primer, NcoI site underlined;SEQ ID NO:29) and 5′-GCAAGCTTCTTGACAGACTCATGTAG-3′ (3′ primer, HindIIIsite underlined; SEQ ID NO:30). C. botulinum type A strain was obtainedfrom the American Type Culture Collection (ATCC#19397) and grown underanaerobic conditions in Terrific broth medium. High molecular-weight C.botulinum DNA was isolated as described in Example 11. The integrity andyield of genomic DNA was assessed by comparison with a serial dilutionof uncut lambda DNA after electrophoresis on an agarose gel.

[0675] The gene fragment was cloned by PCR utilizing a proofreadingthermostable DNA polymerase (native Pfu polymerase). PCR amplificationwas performed using the above primer pair in a 50 μl reaction containing10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 200 μM each dNTP, 0.2μM each primer, and 50 ng C. botulinum genomic DNA. Reactions wereoverlaid with 100 μl mineral oil, heated to 94° C. 4 min, 0.5 μl nativePfu polymerase (Stratagene) was added, and thirty cycles comprising 94°C. for 1 min, 50° C. for 2 min, 72° C. for 2 min were carried outfollowed by 10 min at 72° C. An aliquot (10 μl) of the reaction mixturewas resolved on an agarose gel and the amplified native C fragment genewas gel purified using the Prep-A-Gene kit (BioRad) and ligated topCRScript vector DNA (Stratagene). Recombinant clones were isolated andconfirmed by restriction digestion, using standard recombinant molecularbiology techniques [Sambrook et al. (1989), supra]. In addition, thesequence of approximately 300 bases located at the 5′ end of the Cfragment coding region were obtained using standard DNA sequencingmethods. The sequence obtained was identical to that of the publishedsequence.

[0676] An expression vector containing the native C. botulinum serotypeA C fragment gene was created by ligation of the NcoI-HindIII fragmentcontaining the C fragment gene from the pCRScript clone to NheI-HindIIIrestricted pETHisa vector (Example 18b). The NcoI and NheI sites werefilled in using the Klenow enzyme prior to ligation; these sites werethus blunt-end ligated together. The resulting construct was termedpHisBotA (native). pHisBotA (native) expresses the C. botulinum serotypeA C fragment with a his-tagged N terminal extension which has thefollowing sequence:MetGlyHisHisHisHisHisHisHisHisHisHisSerSerGlyHisIleGluGlyArgHisMetAla(SEQ ID NO:24), where the underlining represents amino acids encoded bythe C. botulinum C fragment gene (this N terminal extension contains therecognition site for Factor Xa protease, shown in italics, which can beemployed to removed the polyhistdine tract from the N-terminus of thefusion protein). The pHisBot (native) construct expresses the identicalprotein as the pHisBot construct (Ex. 24c; herein after the pHisBotA)which contains the synthetic gene.

[0677] The predicted DNA sequence encoding the native C. botulinumserotype A C fragment gene contained within pHisBotA (native) is listedin SEQ ID NO:3′ [the start of translation (ATG) is located atnucleotides 108-110 and the stop of translation (TAA) is located atnucleotides 1494-1496 in SEQ ID NO:31] and the corresponding amino acidsequence is listed in SEQ ID NO:26 (i.e., the same amino acid sequenceas that produced by pHisBotA containing synthetic gene sequences).

[0678] b) Comparison of the Expression and Purification Yields of C.botulinum Serotype A C Fragments Derived from Native and SyntheticExpression Vectors

[0679] Recombinant plasmids containing either the native or thesynthetic C. botulinum serotype A C fragment genes were transformed intoE. coli strain Bl21(DE3) pLysS and protein expression was induced in 1liter shaker flask cultures. Total protein extracts were isolated,resolved on SDS-PAGE gels and C. botulinum C fragment protein wasidentified by Western analysis utilizing a chicken anti-C. botulinumserotype A toxoid antiserum as described in Example 22.

[0680] Briefly, 1 liter (2×YT+100 μg/ml ampicillin and 34 μg/mlchloramphenicol) cultures of bacteria harboring either the pHisBotA(synthetic) or pHisBotA (native) plasmids in the Bl21(DE3) pLysS strainwere induced to express recombinant protein by addition of IPTG to 1 mM.Cultures were grown at 30-32° C., IPTG was added when the cell densityreached an OD₆₀₀ 0.5-1.0 and the induced protein was allowed toaccumulate for 3-4 hrs after induction.

[0681] The cells were cooled for 15 min in a ice water bath and thencentrifuged for 10 min at 5000 rpm in a JAIO rotor (Beckman) at 4° C.The cell pellets were resuspended in a total volume of 40 mls 1× bindingbuffer (40 mM imidazole, 0.5 M NaCl, 50 mM NaPO₄, pH 8.0), transferredto two 50 ml Oakridge tubes and frozen at −70° C. for at least 1 hr. Thetubes were then thawed and the cells were lysed by sonication (usingfour successive 20 second bursts) on ice. The suspension was clarifiedby centrifugation 20-30 min at 9,000 rpm (10,000 g) in a JA-17 rotor.The soluble lysate was batch absorbed to 7 ml of a 1:1 slurry of NiNTAresin:binding buffer by stirring 2-4 hr at 4° C. The slurry wascentrifuged for 1 min at 500 g in 50 ml tube (Falcon), resuspended in 5mls binding buffer and poured into a 2.5 cm diameter column (BioRad).The column was attached to a UV monitor (ISCO) and the column was washedwith binding buffer until a baseline was established Imidazole wasremoved by washing with 50 mM NaPO₄, 0.3 M NaCl, 10% glycerol, pH 7.0and bound protein was eluted using 50 mM NaPO₄, 0.3 M NaCl, 10%glycerol, pH 3.5-4.0.

[0682] The eluted proteins were stored at 4° C. Samples of total,soluble, and eluted proteins were resolved by SDS-PAGE. Protein sampleswere prepared for electrophoresis by mixing 1 μl total (T) or soluble(S) protein with 4 μl PBS and 5 μl 2× SDS-PAGE sample buffer, or 5 μleluted (E) protein and 5 μl 2× SDS-PAGE sample buffer. The samples wereheated to 95° C. for 5 min, then cooled and 5 or 10 μls were loaded on12.5% SDS-PAGE gels. Broad range molecular weight protein markers(BioRad) were also loaded to allow the MW of the identified fusionproteins to be estimated. After electrophoresis, protein was detectedeither generally by staining gels with Coomassie blue, or specifically,by blotting to nitrocellulose for Western blot detection of specificimmunoreactive protein.

[0683] For Western blot analysis, the gels were blotted, and proteintransfer was confirmed by Ponceau S staining as described in Example 22.After blocking the blots for 1 hr at room temperature in blocking buffer(PBST and 5% milk), 10 ml of a {fraction (1/500)} dilution of an anti-C.botulinum toxin A IgY PEG prep (Ex. 3) in blocking buffer was added andthe blots were incubated for an additional hour at room temperature. Theblots were washed and developed using a rabbit anti-chicken alkalinephosphatase conjugate (Boehringer Mannheim) as the secondary antibody asdescribed in Ex. 22. This analysis detected C. botulinum toxinA-reactive proteins in the pHisBotA (native and synthetic) proteinsamples (corresponding to the predicted full length proteins identifiedby Coomassie staining).

[0684] A gel containing proteins expressed from the pHisBot and pHisBot(native) constructs during various stages of purification and stainedwith Coomassie blue is shown in FIG. 31. In FIG. 31, lanes 1-4 and 9contain proteins expressed by the pHisBotA construct (i.e., thesynthetic gene) and lanes 5-8 contain proteins expressed by the pHisBotA(native) construct. Lanes 1 and 5 contain total protein extracts; lanes2 and 6 contain soluble protein extracts; lanes 3 and 7 contain proteinswhich flowed through the NiNTA columns; lanes 4, 8 and 9 contain proteineluted from the NiNTA columns and lane 10 contains molecular weightmarkers.

[0685] The above purification resulted in a yield of 3 mg (native gene)or 11 mg (synthetic gene) of affinity purified protein from a 1 literstarting culture, of which at least 90-95% of the protein was a singleband of the predicted MW (50kd) and immunoreactivity for recombinant C.botulinum serotype A C fragment protein. Other than the level ofexpression, no difference was observed between the native and thesynthetic gene expression systems.

[0686] These results demonstrate that soluble C. botulinum serotype A Cfragment protein can be expressed in E. coli and purified utilizingeither native or synthetic gene sequences.

EXAMPLE 29 Generation of Neutralizing Antibodies Using a Recombinant C.botulinum Serotype A C Fragment Protein Containing a Six Residue His-Tag

[0687] In Example 27, neutralizing antibodies were generated utilizingthe pHisBotA protein, which contains a histidine-tagged N-terminalextension comprising 10 histidine residues. To determine if thegeneration of neutralizing antibodies is dependent on the presence ofthis particular his-tag, a protein containing a shorter N-terminalextension (comprising 6 histidine residues) was produced and tested forthe ability to generate neutralizing antibodies. This example involveda) the cloning and expression of the p6HisBotA(syn) protein and b) thegeneration and characterization of hyperimmune serum.

[0688] a) Cloning and Expression of the p6HisBotA(syn) Protein

[0689] The p6HisBotA(syn) construct was generated as described below;the term “syn” designates the presence of synthetic gene sequences. Thisconstruct expresses the C frgament of the C. botulinum serotype A toxinwith a histidine-tagged N terminal extension having the followingsequence: MetHisHisHisHisHisHisMetAla (SEQ ID NO:32); the amino acidsencoded by the botulinal C fragment gene are underlined and the vectorencoded amino acids are presented in plain type.

[0690] 6×His oligonucleotides [5′-TATGCATCACCATCACCATCA-3′ (SEQ IDNO:33) and 5′-CATGTGATGGTGATGGTGATGCA-3′ (SEQ ID NO:34) were annealed asfollows. One microgram of each oligonucleotide was mixed in total of 20μL 1× reaction buffer 2 (NEB) and the mixture was heated at 70° C. for 5min and then incubated at 42° C. for 5 min. The annealedoligonucleotides were then ligated with gel purified NdeI/HindIIIcleaved pET23b (T7 promoter) or pET21b (T7lac promoter) DNA and the gelpurified NcoI/HindIII C. botulinum serotype A C fragment synthetic genefragment derived from pAlterBot (Ex. 22). Recombinant clones wereisolated and confirmed by restriction digestion. The DNA sequenceencoding the 6×his-tagged BotA protein contained within p6HisBotA(syn)is listed in SEQ ID NO:35. The amino acid sequence of the p6×HisBotAprotein is listed in SEQ ID NO:36.

[0691] The resulting recombinant p6×HisBotA plasmid was transformed intothe BL21(DE3) pLysS strain, and 1 liter cultures were grown, induced andharvested as described in Example 28. His-tagged protein was purified asdescribed in Example 28, with the following modifications. The bindingbuffer (BB) contained 5 mM imidazole rather than 40 mM imidazole andNP40 was added to the soluble lysate to a final concentration of 0.1%.The bound material was washed on the column with BB until the baselinewas established, then the column was washed successively with BB+20 μMimidazole and BB+40 mM imidazole. The column was eluted as described inExample 28.

[0692] In the case of the pET23-derived expression system, high levelexpression of insoluble 6HisBotA protein was induced. The pET21-derivedvector expressed lower levels of soluble protein that bound the NiNTAresin and eluted in the 40 mM imidazole wash rather than during the lowpH elution. These results (i.e., low level expression of a solubleprotein) are consistent with the results obtained with pHisBotA protein(Ex. 25); the pHisBotA construct, like the pET21-derived vector,contains the T7lac rather than T7 promoter.

[0693] The 6HisBotA protein thus elutes under less stringent conditionsthan the 10× histidine-containing pHisBot protein (100-200 mM imidazole;Ex. 25) presumably due to the reduction in the length of the his-tag.The eluted protein was of the predicted size [i.e., slightly reduced incomparison to pHisBotA protein].

[0694] b) Generation and Characterization of Hyperimmune Serum

[0695] Eight BALBc mice were immunized with purified 6HisBotA proteinusing Gerbu GMDP adjuvant (CC Biotech). The 40 mM imidazole elution wasmixed with Gerbu adjuvant and used to immunize mice. Each mouse receiveda subcutaneous injection of 100 μl antigen/adjuvant mix (12 μg antigen+1μg adjuvant) on day 0. Mice were subcutaneously boosted as above on day14 and bled on day 28. Control mice received pHisBotB protein (preparedas described in Ex. 35 below) in Gerbu adjuvant.

[0696] Anti-C. botulinum serotype A toxoid titers were determined inserum from individual mice from each group using the ELISA described inExample 23a with the exception that the initial testing serum dilutionwas 1:100 in blocking buffer containing 0.5% Tween 20, followed byserial 5-fold dilutions into this buffer. The results of the ELISAdemonstrated that seroconversion (relative to control mice) occurred inall 8 mice.

[0697] The ability of the anti-C. botulinum serotype A C fragmentantibodies present in serum from the immunized mice to neutralize nativeC. botulinum type A toxin was tested using the mouse neutralizationassay described in Example 23b. The amount of neutralizing antibodiespresent in the serum of the immunized mice was determined using serumantibody titrations. The various serum dilutions (0.01 ml) were mixedwith 5 LD₅₀ units of C. botulinum type A toxin and the mixtures wereinjected IP into mice. The neutralizations were performed in duplicate.The mice were then observed for signs of botulism for 4 days. Undilutedserum was found to protect 100% of the injected mice while the 1:10diluted serum did not. This corresponds to a neutralization titer of0.05-0.5 IU/ml.

[0698] These results demonstrate that neutralizing antibodies wereinduced when the 6HisBotA protein was utilized as the immunogen.Furthermore, these results demonstrate that seroconversion and thegeneration of neutralizing antibodies does not depend on the specific Nterminal extension present on the recombinant C. botulinum type A Cfragment proteins.

EXAMPLE 30 Construction of Vectors for the Expression of His-Tagged C.botulinum Type A Toxin C Fragment Protein Using the Synthetic Gene

[0699] A number of expression vectors were constructed which containedthe synthetic C. botulinum type A toxin C fragment gene. Theseconstructs vary as to the promoter (T7 or T7lac) and repressor elements(lacIq) present on the plasmid. The T7 promoter is a stronger promoterthan is the T7lac promoter. The various constructs provide varyingexpression levels and varying levels of plasmid stability. This exampleinvolved a) the construction of expression vectors containing thesynthetic C. botulinum type A C fragment gene and b) the determinationof the expression level achieved using plasmids containing either thekanamycin resistance or the ampicillin resistance genes in small scalecultures.

[0700] a) Construction of Expression Vectors Containing the Synthetic C.botulinum Type A C Fragment Gene

[0701] Expression vectors containing the synthetic C. botulinum type A Cfragment gene were engineered to utilize the kanamycin resistance ratherthan the ampicillin resistance gene. This was done for several reasonsincluding concerns regarding the presence of residual ampicillin inrecombinant protein derived from plasmids containing the ampicillinresistance gene. In addition, ampicillin resistant plasmids are moredifficult to maintain in culture; the β-lactamase secreted by cellscontaining ampicillin resistant plasmids rapidly degrades extracellularampicillin, allowing the growth of plasmid-negative cells.

[0702] A second altered feature of the expression vectors is theinclusion of lacIq gene in the plasmid. This repressor lowers expressionfrom lac regulated promoters (the chromosomally located, lactoseregulated T7 polymerase gene and the plasmid located T7lac promoter).This down regulates uninduced protein expression and can enhance thestability of recombinant cell lines. The final alteration to the vectorsis the inclusion of either the T7 or T7lac promoters that drive high ormoderate level expression of recombinant protein, respectively.

[0703] The expression plasmids were constructed as follows. In allcases, the protein expressed is the pHisBotA(syn) protein previouslydescribed, and the only differences between constructs is the alterationof the various regulatory elements described above.

[0704] i) Construction of pHisBotA(syn) kan T7lac The pHisBotA(syn) kanT7lac construct was made by inserting the SapI/XhoI fragment containingthe C. botulinum type A C fragment from pHisBotA(syn) into pET24digested with SapI/XhoI (Novagen; fragment contains kan gene and originof replication). The desired construct was selected for kanamycinresistance and confirmed by restriction digestion.

[0705] ii) Construction Of pHisBotA(syn) kan lacIq T7lac

[0706] The pHisBotA(syn) kan lacIq T7lac construct was made by insertingthe XbaI/HindIII fragment containing the C. botulinum type A C fragmentfrom pHisBotA(syn)kanT7lac into the pET24a vector digested withXbaI/HindIII. The resulting construct was confirmed by restrictiondigestion.

[0707] iii) Construction of pHisBotA(syn) kan lacIq T7

[0708] The pHisBotA(syn) kan lacIq T7 construct was made by insertingthe XbaI/HindIII fragment containing the C. botulinum type A C fragmentfrom pHisBotA(syn) kan lacIq T7lac into XbaI/HindIII-digestedpHisBotB(syn) kan lacIq T7 (described in Ex 37c below). The resultingconstruct was confirmed by restriction digestion.

[0709] b) Determination of the Expression Level Achieved Using PlasmidsContaining Either the Kanamycin Resistance or the Ampicillin ResistanceGenes in Small Scale Cultures

[0710] One liter cultures of pHisBotA(syn) kan T7lac/Bl21(DE3)pLysS andpHisBotA(syn) amp T7lac/Bl21(DE3)pLysS [this is the previouslydesignated pHisBotA(syn) construct] were grown, induced and his-taggedproteins were purified as described in Example 28. No differences inyield or protein integrity/purity were observed.

[0711] These results demonstrate that the antigen induction levels fromexpression constructs were not affected by the choice of ampicillinversus kanamycin antibiotic resistance genes.

EXAMPLE 31 Fermentation of Cells Expressing Recombinant BotulinalProteins

[0712] a) Fermentation Culture of Cells Expressing Recombinant BotulinalProteins

[0713] Fermentation cultures were grown under the following conditionswhich were optimized for growth of the BL21(DE3) strains containing pETderived expression vectors. An overnight 1 liter feeder culture wasprepared by inoculating of 1 liter media (in a 2L shaker flask) with afresh colony grown on an LB kan plate. The feeder culture contained: 600mls nitrogen source [20 gm yeast extract (BBL) and 40 gm tryptone(BBL)/600 mls], 200 mls 5×fermentation salts (per liter: 48.5 gm K₂HPO₄,12 gm NaH₂PO₄H₂O, 5 gm NH₄Cl, 2.5 gm NaCl), 180 mls dH₂O, 20 mls 20%glucose, 2 mls 1 M MgSO₄, 5 mls 0.05M CaCl₂ and 4 mls of a 10 mg/mlkanamycin stock. All solutions were sterilized by autoclaving, exceptthe kanamycin stock which was filter sterilized.

[0714] An aliquot (5 ml) of the feeder culture broth was removed priorto inoculation, and grown for 2 days at 37° C. as a culture brothsterility control. Growth was not observed in this control culture inany of the fermentations performed.

[0715] The inoculated feeder culture was grown for 12-15 hrs (ON) at30-37° C. Care was taken to prevent oversaturation of this culture. Thesaturated feeder culture was added to 10L of fermentation media infermenter (BiofloIV, New Brunswick Scientific, Edison, N.J.) as follows.The fermenter was sterilized 120 min at 121° C. with dH₂O. The sterilewater was removed, and fermentation media added as follows: 6 litersnitrogen source, 2 liters 5× fermentation salts, 2 liters 2% glucose, 20mls 1 M MgSO₄, 50 mls 0.05 M CaCl₂, 2.5-3.5 mls Macol P 400 antifoam(PPG Industries Inc., Gurnee, Ill.), 40 mls 10 mg/ml kanamycin and 10mls trace elements (8 gm FeSO₄.7H₂O, 2 gm MnSO₄.H₂O, 2 gm AlCl₃.6H₂O,0.8 gm CoCl.6H₂O, 0.4 gm ZnSO₄.7H₂O, 0.4 gm Na₂MoO₄.2H₂O, 0.2 gmCuCl₂.2H₂O, 0.2 gm NiCl₂, 0.1 gm H₃BO₄/200 mls 5 M HCl). All solutionswere sterilized by autoclaving, except the kanamycin stock which wasfilter sterilized. Fermentation media was prewarmed to 37° C. before theaddition of the feeder culture.

[0716] After the addition of the feeder culture, the culture wasfermented at 37° C., 400 rpm agitation, and 10 l/min air sparging. TheDO₂ control was set to 20% PID and dissolved oxygen levels werecontrolled by increasing the rate of agitation from 400-850 rpm underDO₂ control. DO₂ levels were maintained at greater than or equal to 20%throughout the entire fermentation. When agitation levels reached500-600 rpm the temperature was lowered to 30° C. to reduce the oxygenconsumption rate. Culture growth was continued until endogenous carbonsources were depleted. In these fermentations, glucose was depletedfirst [monitored with a glucose monitoring kit (Sigma)], followed byassimilation of acetate and other acidic carbons [monitored using anacetate test kit (Boehringer Mannheim)]. During the assimilation phase,the pH rose from 6.6-6.8 (starting pH) to 7.4-7.5, at which time thebulk of the remaining carbon source was depleted. This was signaled by adrop in agitation rate (from a maximum of 700-800 rpm) and a rise in DO₂levels >30%. This corresponds to a OD₆₀₀ reading of 18-20/ml. At thispoint a fed batch mode was initiated, in which a feed solution of 50%glucose was added at a rate of approximately 4 gm glucose/liter/hr. ThepH was adjusted to 7.0 by the addition of 25% H₃PO₄ (approximately 60mls). Culture growth was continued and reached peak oxygen consumptionwithin the next 3 hrs of growth (while the remaining residualnon-glucose carbon sources were assimilated). This phase ischaracterized by a slow increase in pH, and air sparging was increasedto 1SL/min, to keep the maximum rpm below 850.

[0717] Once the residual acidic carbon sources are depleted theagitation rate decreases to 650-750 rpm and the pH begins to drop. pHcontrol was maintained at 7.0 PID by regulated pump addition of asterile 4M NaOH solution which was consumed at a steady rate for theremainder of the fermentation. Growth was continued at 30° C., and thecultures were grown linearly at a growth rate of 4-7 OD₆₀₀ units/hr, toat least 81.5 OD₆₀₀ units/ml (>30 g/l dry cell weight) withoutinduction. Antifoam (a 1:1 dilution with filter sterilized 100% ethanol)was added as necessary throughout the fermentation to prevent foaming.

[0718] During the fed batch mode, glucose was assimilated immediately(concentration in media consistently less than 0.1 gm/liter) and acetatewas not produced in significant levels by the pET plasmid/BL21(DE3) celllines tested (approximately 1 gm/liter at end of fermentation; this islower than that observed in harvests from shaker flask culturesutilizing the same strains). This was fortuitous, since high levels ofacetate has been shown to inhibit induction levels in a variety ofexpression systems. The above described conditions were found to behighly reproducible between fermentations and utilizing differentexpression plasmids. As a result, glucose and acetate level monitoringwere no longer preformed during fermentation.

[0719] b) Induction of Fermentation Cultures

[0720] Induction with IPTG (250 mg-10 gms, depending on the expressionvector and experiment) was initiated 1-3 hrs after initiation of theglucose feed (30-50 OD₆₀₀/ml). The growth rate after induction wasmonitored on a hourly basis. Aliquots (5-10 ml) of cells were harvestedat the time of induction, and at hourly intervals post-induction.Optical density readings were determined by measuring the absorbance at600 nm of 10 μl culture in 990 μl PBS versus a PBS control. The growthrate after induction was found to vary depending on the expressionsystem utilized.

[0721] c) Monitoring of Fermentation Cultures

[0722] Fermentation cultures were monitored using the following controlassays.

[0723] i) Colony Forming Ability

[0724] An aliquots of cells were removed from the cultures at eachtimepoint sampled (uninduced and at various times after induction) wereserially diluted in PBS (dilution 1=15 μl cells/3 ml PBS, dilution 2=15μl of dilution ⅓ ml PBS, dilution 3=3 or 6 μl of dilution ⅔ mls PBS) and100 μl of dilution 3 was plated on an LB or TSA (trypticase soy agar)plate. The plates were incubated ON at 37° C. and then the colonies arecounted and scored for macro or micro growth.

[0725] ii) Phenotypic Characterization

[0726] Colonies growing on LB or TSA plates (above) from uninduced andinduced timepoints were replica plated onto LB+kan, LB+chloramphenicol(for fermentations utilizing LysS or pACYCGro plasmids), LB+kan+1 mMIPTG and LB plates, in this order. The plates were grown 6-8 hrs at 37°C. and growth was scored on each plate for a minimum of 40-50 wellisolated colonies. The percentage of cells retaining the plasmid at timeof induction (i.e., uninduced cultures immediately prior to the additionof IPTG) was determined to be the # colonies LB+Kan (orchlorarnphenicol) plate/# colonies LB plate×100%. The percentage ofcells with mutated pET plasmids was determined to be the # coloniesLB+Kan+IPTG plate/# colonies LB plate×100%. Colonies on all LB plateswere scored morphologically for E. coli phenotype as a contaminationcontrol. Morphologically detectable contaminant colonies were notdetected in any fermentation.

[0727] iii) Recombinant BotA Protein Induction

[0728] A total of 10 OD₆₀₀₀ units of cells (e.g., 200 μl of cells atOD₆₀₀=50/ml) were removed from each timepoint sample to a 1.5 mlmicrofuge tube and pelleted for 2 min at maximum rpm in a microfuge. Thepellets were resuspended in 1 ml of 50 mM NaHPO₄, 0.5 M NaCl, 40 mMimidazole buffer (pH 6.8) containing 1 mg/ml lysozyme. The samples wereincubated for 20 min at room temperature and stored ON at −70° C.Samples were thawed completely at room temperature and sonicated 2×10seconds with a Branson Sonifier 450 microtip probe at # 3 power setting.The samples were centrifuged for 5 min at maximum rpm in a microfuge.

[0729] An aliquot (20 μl) of the protein samples were removed to 20 μl2× sample buffer, before or after centrifugation, for total and solubleprotein extracts, respectively. The samples were heated to 95° C. for 5min, then cooled and 5 or 10 μl were loaded onto 12.5% SDS-PAGE gels.High molecular weight protein markers (BioRad) were also loaded to allowfor estimation of the MW of identified fusion proteins. Afterelectrophoresis, protein was detected either generally by staining gelswith Coomassie blue, or specifically, by blotting onto nitrocellulose(as described in Ex. 28) for Western blot detection of specifichis-tagged proteins utilizing a NiNTA-alkaline phosphatase conjugateexactly as described by the manufacturer (Qiagen).

[0730] iv) Recombinant Antigen Purification

[0731] At the end of each fermentation run, 1-10 liters of culture wereharvested from the fermenter and the bacterial cells were pelleted bycentrifugation at 6000 rpm for 10 min in a JAIO rotor (Beckman). Thecell pellets were stored frozen at −70° C. or utilized immediatelywithout freezing. Cell pellets were resuspended to 15-20% weight tovolume in resuspension buffer (generally 50 mM NaPO₄, 0.5 M NaCl, 40 mMimidazole, pH 6.8) and lysed utilizing either sonication or highpressure homogenization.

[0732] For sonication, the resuspension buffer was supplemented withlysozyme to 1 mg/ml, and the suspension was incubated for 20 min. atroom temp. The sample was then frozen ON at −70° C., thawed andsonicated 4×20 seconds at microtip maximum to reduce viscosity. Forhomogenization, the cells were lyzed by 2 passes through a homogenizer(Rannie Mini-lab type 8.30 H) at 600 Bar. Cell lysates were clarified bycentrifugation for 30 min at 10,000 rpm in a JA10 rotor.

[0733] For IDA chromatography, samples were flocculated utilizingpolyethyleneimine (PEI) prior to centrifugation. Cell pellets wereresuspended in cell resuspension buffer (CRB: 50 mM NaPO₄, 0.5 M NaCl,40 mM imidazole, pH 6.8) to create a 20% cell suspension (wet weight ofcells/volume of CRB) and cell lysates were prepared as described above(sonication or homogenization). PEI (a 2% solution in dH₂O, pH 7.5 withHCl) was added to the cell lysate a final concentration of 0.2%, andstirred for 20 min at room temperature prior to centrifugation (8,500rpm in JAIO rotor for 30 minutes at 4° C.). This treatment removed RNA,DNA and cell wall components, resulting in a clarified, low viscositylysate (“PEI clarified lysate”).

[0734] His-tagged proteins were purified from soluble lysates bymetal-chelate affinity chromatography using either a NiNTA resin (asdescribed in Ex. 28) or an IDA (iminodiacetic acid) resin as describedbelow.

[0735] IDA resin affinity purifications were performed utilizing a lowpressure chromatography system (ISCO). A 7 ml (small scale) or 70 ml(large scale) Chelating Sepharose Fast Flow (Pharmacia) affinity columnwas poured; in addition, a second guard column was poured and attachedin line with the first column (to capture Ni ions that leached off theaffinity column). The columns were washed with 3 column volumes of dH₂O.The guard column was then removed and the affinity column was washedwith 0.3 M NiSO₄ until resistivity was established, then with dH₂O untilthe resistivity returned to baseline. The columns were reconnected andequilibrated with cell resuspension buffer (CRB; 50 mM NaPO₄, 0.5 MNaCl, 40 mM imidazole, pH 6.8). The clarified sample (in CRB) wasloaded. Flow rates were 5 ml/min for small scale columns and 20 m/minfor large scale columns. After sample loading, the column was washedwith CRB until a baseline established and bound protein was eluted withelution buffer (50 mM NaPO₄, 0.5 M NaCl, 800 mM imidazole, 20% glycerol,pH 6.8 or 8.0). Protein samples were stored at 4° C. or −20° C. Theyield of eluted protein was established by measuring the OD₂₈₀ of theelutions, with a 1 mg/ml solution of protein assumed to yield anabsorbance reading of 2.0.

[0736] The IDA columns may be regenerated and reused multiple times(>10). To regenerate the column, the column was washed with 2-3 columnvolumes of H₂O, then 0.05 M EDTA until all of the blue/green color wasremoved followed by a wash with dH₂O. The IDA columns were sterilizedwith 0.1 M NaOH (using at least 3 column volumes but not more than 50minutes contact time with column packing material), then washed with 3column volumes 0.05 M NaPO₄, pH 5.0, then dH₂O and stored at roomtemperature in 20% ethanol.

EXAMPLE 32 Construction of a Folding Chaperone Overexpression System

[0737] Co-overexpression of the E. coli GroEL/GroES folding chaperonesin a cell expressing a recombinant foreign protein has been reported toenhance the solubility of some foreign proteins that are otherwiseinsoluble when expressed in E coli [Gragerouu et al. (1992) Proc. Natl.Acad. Sci. USA 89:10344]. The improvement in solubility is thought to bedue to chaperone-mediated binding and unfolding of insoluble denaturedproteins, thus allowing multiple attempts for productive refolding ofrecombinant proteins. By overexpressing the chaperones, theunfolding/refolding reaction is driven by excess chaperone, resulting,in some cases, in higher yields of soluble protein.

[0738] In this example, a chaperone overexpression system, compatiblewith pET vector expression systems, was constructed to facilitatetesting chaperone-mediated solubilization of C. botulinum type Aproteins. This example involved the cloning of the GroEL/ES operon andconstruction of a pLysS-based chaperone hyperexpression system.

[0739] The GroEL/GroES operon was PCR amplified and cloned into thepCRScript vector as described in Example 28. The following primer pairwas used: 5′-CGCAT ATGAATATTCGTCCATTGCATG-3′ (SEQ ID NO:37) [5′ primer,start codon of groES gene converted to NdeI site (underlined)] and5′-GGAAGCTTGCAGGGCAAT TACATCATG (SEQ ID NO:38) (3′ primer, stop codon ofgroEL gene italicized, engineered HindIII site underlined). Followingamplification, the chaperone operon was excised as an NdeI/HindIIIfragment and cloned into pET23b digested with NdeI and HindIII. Thisconstruction places the Gro operon under the control of the T7 promoterof the pET23 vector. The desired construct was confirmed by restrictiondigestion.

[0740] The T7 promoter-Gro operon-T7 terminator expression cassette wasthen excised as a BglII/BspEI (filled) fragment and cloned into BamHI(compatible with BglII)/HindIII (filled) cleaved pLysS plasmid (thisremoved the T7 lysozyme gene). The resulting construct was designatedpACYCGro, since the plasmid utilizing the pACYC184 origin from the plysSplasmid. Proper construction was confirmed by restriction digestion.

[0741] pACYCGro was transformed into BL21(DE3), cultures were grown andinduced with 1 mM IPTG as described in preceding examples. Total andsoluble protein extracts were generated from cells removed before andafter IPTG induction and were resolved on a 12.5% SDS-PAGE gel andstained with Coomassie blue. This analysis revealed that high levels ofsoluble GroEI and GroES proteins were made in the induced cells. Theseresults demonstrated that the chaperone hyper-expression system wasfunctional.

EXAMPLE 33 Growth of BotA/pACYCGro Cell Lines in Fermentation Cultures

[0742] Induction of BL21(DE3) cells lacking the LysS plasmid whichcontained BotA expression constructs grown in shaker flask orfermentation culture resulted in the expression of primarily insolubleBotA protein. Fermentation cultures were performed to determine if thesimultaneous overexpression of the Gro operon and recombinant C.botulinum type A proteins (BotA proteins) resulted in enhancedsolubility of the recombinant BotA protein. This example involved thefermentation of pHisBotA(syn)kan lacIq T7lac/pACYCGro BL21(DE3) andpHisBotA(syn)kan lacIq T7/pACYCGro BL21(DE3) cell lines. Thefermentations were repeated exactly as described in Example 31.Chloramphenicol (34 μg/ml) was included in the feeder and fermentationcultures.

[0743] a) Fermentation of pHisBotA(syn)kan lacIq T7lac/pACYCGroBL21(DE3) Cells

[0744] For fermentation of cells containing plasmids comprising theT7lac promoter, induction was with 2 gms IPTG at 1 hr post initiation ofglucose feed. The OD₆₀₀ was 35 at time of induction, then 48.5, 61.5, 67at 1-3 hrs post induction. Viable colony counts decreased from 0-3 hrinduction [21 (13), 0, 0, 0; dilution 3 utilized 3 μl of dilution 2cells] with numbers in parenthesis for the indicating microcolonies. Of28 colonies scored at the time of induction, 23 retained thepHisBotA(syn)kan lacIq T7lac plasmid (kan resistant), 22 contained thechaperone plasmid (chloramphenicol resistant) and no colonies atinduction grew on IPTG+Kan plates (no mutations detected). These resultswere indicative of very strong promoter induction, since colonyviability dropped immediately after induction.

[0745] Total and soluble extracts were resolved on a 12.5% SDS-PAGE geland stained with Coomassie. High level induction of Gro chaperones wasobserved, but very low level expression of soluble BotA protein wasobserved, increasing from 1 to 4.0 hrs post induction (no expressiondetected in uninduced cells). The dramatically lower expression of theBotA antigen in the presence of chaperone may be due to promoterocclusion (i.e., the stronger T7 promoter on the chaperone plasmid ispreferentially utilized).

[0746] b) Fermentation of pHisBotA(syn)kan lacIq T7/pACYCGro BL21(DE3)Cells

[0747] A fermentation utilizing the T7-driven BotA expression plasmidwas performed. Induction was with 1 gm IPTG at 2 hrs post initiation ofglucose feed. The OD₆₀₀ was 41 at time of induction, then 51.5, 61.5,61.5 and 66 at 1-4 hrs post induction. Viable colony counts decreasedfrom 0-4 hrs induction [71, 1 (34), 1 (1), 1, 0; dilution 3 utilized 6μl dilution 2 cells) with numbers in parenthesis for the uninducedtimepoint indicating microcolonies. Of 65 colonies scored at the time ofinduction, all 65 retained both the pHisBotA(syn)kan lacIq T7 plasmid(kan resistant) and the chaperone plasmid (chloramphenicol resistant)and no colonies at induction grew on IPTG+Kan plates (no mutationsdetected).

[0748] Total and soluble extracts were resolved on a 12.5% SDS-PAGE geland stained with Coomassie. High level induction of Gro chaperones andmoderate level expression of soluble BotA protein was observed,increasing from 1 to 4.0 hrs post induction (no expression detected inuninduced cells).

[0749] A PEI-clarified lysate (0.2% final cocnentration PEI) [850 mlfrom 130 gm cell pellet (2 liters fermentation harvest)] was purified ona large scale IDA column. A total of 78 mg of protein was eluted.Extracts from the purification were resolved on a 12.5% SDS-PAGE gel andstained with Coomassie. The elution was found to contain anapproximately 1:1 mix of BotA/chaperone protein (FIG. 32). PEI lysatesprepared in this manner were typically 16 OD₂₈₀/ml. This was estimatedto be 8 mg protein/ml of lysate (by BCA assay). Thus, the elutedrecombinant BotA protein represented 0.55% of the total soluble cellularprotein applied to the column.

[0750] In FIG. 32, lane 1 contains molecular weight markers, lanes 2-9contain extracts from pHisBotA(syn)kan lacIq T7/pACYCGro/BL21(DE3) cellsbefore or during purification on the IDA column. Lane 2 contains totalprotein extract; lane 3 contains soluble protein extract; lanes 4 and 5contain PEI-clarified lysates (duplicates); lanes 6 and 7 containflow-through from the IDA column (duplicates) and lanes 8 and 9 containIDA column elute (lane 9 contains {fraction (1/10)} the amount appliedto lane 8).

[0751] These results demonstrate, that although the majority of the BotAprotein produced was insoluble, 20 mg/liter of soluble recombinant BotAprotein can be purified utilizing the pHisBotA(syn)kan lacIqT7/pACYCGro/BL21(DE3) expression system.

EXAMPLE 34 Purification of Recombinant BotA Protein from FoldingChaperones

[0752] In this example of size exclusion chromatography was used topurify the recombinant BotA protein away from the folding chaperones andimidazole present in the IDA-purified material (Ex. 33).

[0753] To enhance the solubility of the recombinant BotA protein duringscale-up, the protein was co-expressed with folding chaperones (Ex. 33).As observed with the recombinant BotB protein (Example 40 below), thefolding chaperones co-eluted with the recombinant BotA protein duringthe Ni-IDA purification step. Because the recombinant BotA and BotBproteins have similar molecular weights (about {fraction (1/10)} thesize of the non-reduced folding chaperone) and the imidazole stepgradient strategy was unsuccessful in purifying BotB away from thefolding chaperone (see Ex. 40), size exclusion chromatography wasexamined for the ability to purify the recombinant BotA protein awayfrom the folding chaperones.

[0754] A column (2.5×24 cm) containing Sephacryl S-100 HR (Pharmacia)was poured (bed volume˜110 ml). Proteins having molecular weightsgreater than 100 K are expected to elute in the void volume under theseconditions and smaller proteins should be retained by the beads andelute at different times, depending on their molecular weights. Tomaintain solubility of the purified BotA protein, the Sephacryl columnwas equilibrated in a buffer having the same salt concentration as thebuffer used to elute the BotA protein from the IDA column (i.e., 50 mMsodium phosphate, 0.5 M NaCl, 10% glycerol; all reagents fromMallinkrodt, Chesterfield, Mo.).

[0755] Five milliliters of the IDA-purified recombinant BotA protein(Ex. 33) was filtered through a 0.45μ syringe filter, applied to thecolumn and the equilibration buffer was pumped through the column at aflow rate of 1 ml/minute. Eluted proteins were monitored by absorbanceat 280 nm and collected either manually or with a fraction collector(BioRad). Appropriate fractions were pooled, if necessary, and theprotein was quantitated by absorbance at 280 nm and/or BCA protein assay(Pierce). The isolated peaks were then analyzed by native and/orSDS-PAGE to identify the proteins present and to evaluate purity. Thefolding chaperone eluted first, followed by the recombinant BotA proteinand then the imidazole peak.

[0756] SDS-PAGE analysis (12.5% polyacrylamide, reduced samples) wasused to evaluate the purity of the IDA-purified recombinant BotA proteinbefore and after S-100 purification. FIG. 33 shows the difference inpurity before and after the S-100 purification step. In FIG. 33, lane 1contains molecular weight markers (BioRad broad range). Lane 2 shows theIDA-purified recombinant BotA protein preparation, which is contaminatedwith significant amounts of the folding chaperone. Following S-100purification, the amount of folding chaperone present in the BotA sampleis reduced dramatically (lane 3). Lane 4 contains no protein (i.e., itis a blank lane); lanes 5-8 contain samples of IDA-purified recombinantBotB and BotE proteins and are discussed infra.

[0757] Endotoxin levels in the S-100 purified BotA preparation weredetermined using the LAL assay (Associates of Cape Cod) as describe inExample 24. The purified BotA preparation was found to contain 22.7 to45.5 EU/mg recombinant protein.

[0758] These results demonstrate that size exclusion chromatography wassuccessful in purifying the recombinant BotA protein from foldingchaperones and imidazole following an initial IDA purification step.Furthermore, these results demonstrate that the S-100 purified BotAprotein was substantially free of endotoxin.

EXAMPLE 35 Cloning and Expression of the C Fragment of the C. botulinumSerotype B Toxin Gene

[0759] The C. botulinum type B neurotoxin gene has been cloned andsequenced [Whelan et al. (1992) Appl. Environ. Microbiol. 58:2345 andHutson et al. (1994) Curr. Microbiol. 28:101]. The nucleotide sequenceof the toxin gene derived from the Eklund 17B strain (ATCC 25765) isavailable from the EMBL/GenBank sequence data banks under the accessionnumber X71343; the nucleotide sequence of the coding region is listed inSEQ ID NO:39. The amino acid sequence of the C. botulinum type Bneurotoxin derived from the strain Eklund 17B is listed in SEQ ID NO:40.The nucleotide sequence of the C. botulinum serotype B toxin genederived from the Danish strain is listed in SEQ ID NO:41 and thecorresponding amino acid sequence is listed in SEQ ID NO:42.

[0760] The DNA sequence encoding the native C. botulinum serotype B Cfragment gene derived from the Eklund 17B strain can be expressed usingthe pETHisb vector; the resulting coding region is listed in SEQ IDNO:43 and the corresponding amino acid sequence is listed in SEQ IDNO:44. The DNA sequence encoding the native C. botulinum serotype B Cfragment gene derived from the Danish strain can be expressed using thepETHisb vector; the resulting coding region is listed in SEQ ID NO:45and the corresponding amino acid sequence is listed in SEQ ID NO:46. TheC frgament region from any strain of C. botulinum serotype B can beamplified and expressed using the approach illustrated below using the Cfragment derived from C. botulinum type B 2017 strain.

[0761] The C. botulinum type B neurotoxin gene is synthesized as asingle polypeptide chain which is processed to form a dimer composed ofa light and a heavy chain linked via disulfide bonds; the type Bneurotoxin has been reported to exist as a mixture of predominatlysingle chain with some double chain (Whelan et al., supra). The 50 kDcarboxy-terminal portion of the heavy chain is referred to as the Cfragment or the H_(C) domain. Expression of the C fragment of C.botulinum type B toxin in heterologous hosts (e.g., E. coli) has notbeen previously reported.

[0762] The native C fragment of the C. botulinum serotype B toxin genewas cloned and expression constructs were made to facilitate proteinexpression in E. coli. This example involved PCR amplification of thegene, cloning, and construction of expression vectors.

[0763] The C fragment of the C. botulinum serotype B (BotB) toxin genewas cloned using the protocols and conditions described in Example 28for the isolation of the native BotA gene. The C. botulinum type B 2017strain was obtained from the American Type Culture Collection (ATCC#17843). The following primer pair was used to amplify the BotB gene:5′-CGCCATGGCTGATACAATACTAATAGAA ATG-3′ [5′ primer, engineered NcoI siteunderlined (SEQ ID NO:47)] and 5′-GCAAG CTTTATTCAGTCCACCCTTCATC-3′ [3′primer, engineered HindIII site underlined, native gene terminationcodon italicized (SEQ ID NO:48)]. After cloning into the pCRscriptvector, the NheI(filled)/HindIII fragment was cloned into pETHisb vectoras described for BotA C fragment gene in Example 28. The resultingconstruct was termed pHisBotB. pHisBotB expresses the BotB genesequences under the transcriptional control of the T7 lac promoter andthe resulting protein contains an N-terminal 10× His-tag affinity tag.The pHisBotB expression construct was transformed into BL21 (DE3) pLysScompetent cells and 1 liter cultures were grown, induced and his-taggedproteins were purified utilizing a NiNTA resin (eluted in low pH elutionbuffer) as described in Example 28. Total, soluble and purified proteinswere resolved by SDS-PAGE and detected by Coomassie staining and Westernblot hybridization utilizing a chicken anti-C. botulinum serotype Btoxoid primary antibody (generated by immunization of hens using C.botulinum serotype B toxoid as described in Example 3). Samples of BotAand BotE C fragment proteins were included on the gels for MW andimmunogenicity comparisons. Strong immunoreactivity to only the BotBprotein was detected with the anti-C. botulinum serotype B toxoidantibodies. The recombinant BotB protein was expressed at low levels (3mg/liter) as a soluble protein. The purified BotB protein migrated as asingle band of the predicted MW (i.e., ˜50 kD).

[0764] These results demonstrate the cloning of the native C. botulinumserotype B C fragment gene, the expression and purification of therecombinant BotB protein as a soluble his-tagged protein in E. coli.

EXAMPLE 36 Generation of Neutralizing Antibodies Using the RecombinantpHisBotB Protein

[0765] The ability of the purified pHisBot protein to generateneutralizing antibodies was examined. Nine BALBc mice were immunizedwith BotB protein (purified as described in Ex. 35) using Gerbu GMDPadjuvant (CC Biotech). The low pH elution was mixed with Gerbu adjuvantand used to immunize mice. Each mouse received a subcutaneous injectionof 100 μl antigen/adjuvant mix (15 μg antigen+1 μg adjuvant) on day 0.Mice were subcutaneously boosted as above on day 14 and bled on day 28.Mice were subsequently boosted 1-2 weeks after bleeding and were thenbled on day 70.

[0766] Anti-C. botulinum serotype B toxoid titers were determined in day28 serum from individual mice from each group using the ELISA protocoloutlined in Example 29 with the exception that the plates were coatedwith C. botulinum serotype B toxoid, and the primary antibody was achicken anti-C. botulinum serotype B toxoid. Seroconversion [relative tocontrol mice immunized with pHisBotE antigen (described below)] wasobserved with all 9 mice immunized with the purified pHisBotB protein.

[0767] The ability of the anti-BotB antibodies to neutralize native C.botulinum type B toxin was tested in a mouse-C. botulinum neutralizationmodel using pooled mouse serum (see Ex. 23b). The LD₅₀ of purified C.botulinum type B toxin complex (Dr. Eric Johnson, University ofWisconsin, Madison) was determined by a intraperitoneal (IP) method[Schantz and Kautler (1978), supra] using 18-22 g female ICR mice. Theamount of neutralizing antibodies present in the serum of the immunizedmice was determined using serum antibody titrations. The various serumdilutions (0.01 ml) were mixed with 5 LD₅₀ units of C. botulinum type Btoxin and the mixtures were injected IP into mice. The neutralizationswere performed in duplicate. The mice were then observed for signs ofbotulism for 4 days. Undiluted serum (day 28 or day 70) was found toprotect 100% of the injected mice while the 1:10 diluted serum did not.This corresponds to a neutralization titer of 0.05-0.5 IU/ml.

[0768] These results demonstrate that seroconversion occurred andneutralizing antibodies were induced when the pHisBotB protein wasutilized as the immunogen.

EXAMPLE 37 Construction of Vectors to Facilitate Expression ofHis-Tagged BotB Protein in Fermentation Cultures

[0769] A number of expression vectors were constructed to facilitate theexpression of recombinant BotB protein in large scale fermentationculture. These constructs varied as to the strength of the promoterutilized (T7 or T7lac) and the presence of repressor elements (lacIq) onthe plasmid. The resulting constructs varied in the level of expressionachieved and in plasmid stability which facilitated the selection of aoptimal expression system for fermentation scaleup.

[0770] The BotB expression vectors created for fermentation culture wereengineered to utilize the kanamycin rather than the ampicillinresistance gene, and contained either the T7 or T7lac promoter, with orwithout the lacIq gene for the reasons outlined in Example 30.

[0771] In all cases, the protein expressed by the various expressionvectors is the pHisBot B protein described in Example 35, with the onlydifferences between clones being the alteration of various regulatoryelements. Using the designations outlined below, the pHisBotB clone (Ex.35) is equivalent to pHisBotB amp T7lac.

[0772] a) Construction of pHisBotB kan T7lac

[0773] pHisBotB kan T7lac was constructed by insertion of theBglII/HindIII fragment of pHisBotB which contains the BotB genesequences into the pPA1870-2680 kan T7lac vector which had been digestedwith BglII and HindIII (the pPA1870-2680 kan T7lac vector contains thepET24 kan gene in the pET23 vector, such that no lacIq gene is present).Proper construction of pHisBotB kan T7lac was confirmed by restrictiondigestion.

[0774] b) Construction of pHisBotB kan lacIq T7lac

[0775] pHisBotB kan lacIq T7lac was constructed by insertion of theBglII/HindIII fragment of pHisBotB which contains the BotB genesequences into similarly cut pET24a vector. Proper construction ofpHisBotB kan lacIq T7lac was confirmed by restriction digestion.

[0776] c) Construction of pHisBotB kan lacIq T7

[0777] pHisBotB kan lacIq T7 was constructed by inserting the NdeI/XhoIfragment from pHisBotE kan lacIq T7lac which contains the BotB genesequences into similarly cleaved pPA1870-2680 kan lacIq T7 vector (thisvector contains the T7 promoter, the same N-terminal his-tag as the Botconstructs, the C. difficile toxin A insert, and the kan lacIq genes;this cloning replaces the C. difficile toxin A insert with the BotBinsert). Proper construction was confirmed by restriction digestion.

[0778] Expression of recombinant BotB protein from these expressionvectors and purification of the BotB protein is described in Example 38below.

EXAMPLE 38 Fermentation and Purification of Recombinant BotB ProteinUtilizing the pHisBotB kan lacIq T7lac, pHisBotB kan T7lac and pHisBotBkan lacIq T7 Vectors

[0779] The pHisBotB kan lacIq T7lac, pHisBotB kan T7lac and BotB kanlacIq T7 constructs [all transformed into the Bl21(DE3) strain] weregrown in fermentation cultures to determine the utility of the variousconstructs for large scale expression and purification of soluble BotBprotein. All fermentations were performed as described in Example 31.

[0780] a) Fermentation of pHisBotB kan lacIq T7lac/Bl21(DE3) Cells

[0781] The fermentation culture was induced 45 min post start of glucosefeed with 1 gm IPTG (final concentration=0.4 mM). pH was maintained at6.5 rather than 7.0. The OD₆₀₀ was 27 at time of induction, then 35, 38,and 40 at 1-3 hrs post induction. Duplicate platings of diluted 1 hrinduction samples (dilutions were prepared as described Ex. 31, dilution3 utilized 3 μl of dilution 2 cells) on TSA and LB+kan plates yielded 89TSA colonies and 81 kan colonies (90% kan resistant).

[0782] Total and soluble protein extracts were resolved on a 12.5%SDS-PAGE gel and total protein was detected by staining with Coomassieblue. Low level induction of insoluble Bot B protein was observed,increasing from 1 to 3 hrs post induction (no expression was detected inuninduced cells).

[0783] b) Fermentation of pHisBotB kan T7lac/Bl21(DE3) Cells

[0784] The fermentation culture was induced 1 hr post start of glucosefeed with 2 gm IPTG (final concentration=0.8 mM). pH was maintained at6.5 rather than 7.0. The OD₆₀₀ was 24.5 at time of induction, then 31.5,32, and 33 at 1-3 hrs post induction, respectively. Duplicate platingsof diluted 0 hr and 2 hr induction samples (dilutions were prepared asdescribed Ex. 31; dilution 3 utilized 3 μl of dilution 2 cells) on TSAand LB+kan plates yielded 32 TSA colonies and 54 kan colonies (all kanresistant) for uninduced cells, and 1 TSA colony and 0 kan colonies 2 hrpost induction. These results were indicative of strong induction, sinceviable counts decreased dramatically 2 hrs post induction.

[0785] Total and soluble extracts were resolved on a 10% SDS-PAGE geland total protein was detected by staining with Coomassie blue. Moderateinduction of insoluble BotB protein was observed, increasing from 1 to 3hrs post induction (no expression was detected in uninduced cells).

[0786] c) Fermentation of pHisBotB kan lacIq T7/Bl21(DE3) Cells

[0787] The fermentation was induced 2 hr post start of glucose feed with4 gm IPTG (final concentration=1.6 mM). pH was maintained at 6.5 ratherthan 7.0. The OD₆₀₀ was 45 at time of induction, then 47, 50, and 50 and55 at 1-4 hrs post induction, respectively. Viable colony countsdecreased after induction (96, 1, 1, 2, 3; dilution 3 utilized 3 μl ofdilution 2 cells). Of 63 colonies scored at the time of induction, all63 retaining the BotB plasmid (kan resistant) and no colonies atinduction grew on IPTG+Kan plates (no mutations detected).

[0788] Total and soluble extracts were resolved on a 12.5% SDS-PAGE geland total protein was detected by staining with Coomassie blue. Moderatelevel induction of insoluble BotB protein was observed, increasing from1 to 4 hrs post induction (lower level expression was detected inuninduced cells, since the T7 rather than T7lac promoter was utilized).

[0789] d) Purification of pHisBotB Protein from pHisBotB ampT7lac/Bl21(DE3) Cells

[0790] Soluble recombinant BotB protein was purified utilizing NiNTAresin from 80 ml of cell lysate generated from cells harvested from apHisBotB fermentation [using the pHisBotB amp T7lac/Bl21(DE3) strain].As predicted from the small scale results above, the majority of theinduced protein was insoluble. As well, the eluted material wascontaminated with multiple E. coli contaminant proteins. A Coomassieblue-stained SDS-PAGE gel containing extracts derived from pHisBotB ampT7lac/Bl21(DE3) cells before and during purification is shown in FIG.34. In FIG. 34, lane 1 contains broad range protein MW markers (BioRad).Lanes 2-5 contain extracts prepared from pHisBotB amp T7lac/Bl21(DE3)cells grown in fermentation culture; lane 2 contains total protein; lane3 contains soluble protein; lane 4 contains protein which did not bindto the NiNTA column (i.e., the flow-through) and lane 5 contains proteineluted from the NiNTA column.

[0791] Similar results were obtained using a small scale IDA columnutilizing a cell lysate from the pHisBotB kan lacIq T7 fermentationdescribed above. 250 mls of a 20% w/v PEI clarified lysate (50 gms cellpellet) of botB kan lacIq T7/Bl21(DE3) cells were purified on a smallscale IDA column. The total yield of eluted protein was 21 mg protein(assuming 1 mg/ml solution=2 OD₂₈₀/ml). When analyzed by SDS-PAGE andCoomassie staining, the BotB protein was found to comprise approximately50% of the eluted protein with the remainder being a ladder of E. coliproteins similar to that observed with the NiNTA purification.

[0792] The NiNTA alkaline phosphatase conjugate was utilized to detecthis-tagged proteins on a Western blot containing total, soluble, soluble(PEI clarified), soluble (after IDA column) and elution samples from theIDA column purification. The results demonstrated that a smallpercentage of BotB protein was soluble, that the soluble protein was notprecipitated by PEI treatment and was quantitatively bound by the IDAcolumn. Since a 1 liter fermentation harvest yielded a 67.5 gm cellpellet, this indicated that the yield of soluble affinity purified BotBprotein from the IDA column was 14 mg/liter.

EXAMPLE 39 Co-Expression of Recombinant BotB Proteins and FoldingChaperones in Fermentation Cultures

[0793] Fermentations were performed to determine if the simultaneousoverexpression of folding chaperones (i.e., the Gro operon) and the BotBprotein resulted in enhanced solubility of the Bot B protein. Thisexample involved fermentation of the pHisBotBkan lacIq T7lac/pACYCGroBL21(DE3), pHisBotB kan T7lac/pACYCGro Bl21(DE3) and pHisBotBkan lacIqT7/pACYCGro BL21(DE3) cell lines. Fermentation was carried out asdescribed in Example 31; 34 μg/ml chloramphenicol was included in thefeeder and fermentation cultures.

[0794] a) Fermentation of pHisBotBkan lacIq

[0795] T7lac/pACYCGro BL21(DE3) Cells Induction was with 4 gms IPTG at 1hr 15 min post initiation of the glucose feed. The OD₆₀₀ was 38 at timeof induction, then 50, 58.5, 62 and 68 at 1-4 hrs post induction. Viablecolony counts decreased during induction (24, 0, 0, 2, 0 at 0-4 hrinduction; dilution 3 utilized 3 μl of dilution 2 cells). Of 24 coloniesscored at the time of induction, 24 retained the BotB plasmid (kanresistant), 24 contained the chaperone plasmid (chloramphenicolresistant) and no colonies at induction grew on IPTG+Kan plates (nomutations detected).

[0796] Total and soluble extracts were resolved on 12.5% SDS-PAGE gelsand were either stained with Coomassie blue or subjected to Westernblotting (his-tagged proteins were detected utilizing the NiNTA-alkalinephosphatase conjugate). This analysis revealed that the Gro chaperoneswere induced to high levels, but very low level expression of solubleBotB protein was observed, increasing from 1 to 4.0 hrs post induction(no expression detected in uninduced cells, induced protein detectedonly on Western blot). The dramatically lower expression of BotB proteinin the presence of chaperone may be due to promoter occlusion (i.e., thestronger T7 promoter on the chaperone plasmid was preferentiallyutilized).

[0797] b) Fermentation of pHisBotB kan T7lac/pACYCGro/Bl21(DE3) Cells

[0798] Induction was with 4 gms IPTG at 1 hr post initiation of theglucose feed. The OD₆₀₀ was 33.5 at time of induction, then 44, 51, 58.5and 69 at 1-4 hrs post induction. Viable colony counts decreased after 2hrs induction (43, 65, 74, 0 (70), 0 (70) at 0-4 hr induction; bracketednumbers represent microcolonies; dilution 3 utilized 3 μl of dilution 2cells). Most colonies at induction retained the BotB plasmid (kanresistant)and the chaperone plasmid (chloramphenicol resistant) and nocolonies at induction grew on IPTG+Kan plates (no mutations detected).

[0799] Total and soluble extracts were resolved on a 12.5% SDS-PAGE geland subjected to Western blotting; his-tagged proteins were detectedutilizing the NiNTA-alkaline phosphatase conjugate. This analysisrevealed that the Gro chaperones were induced to high levels and lowlevel expression of soluble Bot B protein was observed, increasing from1 to 4.0 hrs post induction (no expression detected in uninduced cells).

[0800] A small scale IDA purification of BotB protein from a 250 ml PEIclarified 15% w/v extract (37.5 gm cell pellet) yielded approximately12.5 mg protein, of which approximately 50% was BotB protein and 50% wasGroEL chaperone (assessed by Coomassie staining of a 10% SDS-PAGE gel).The NiNTA alkaline phosphatase conjugate was utilized to detecthis-tagged proteins on a Western blot containing total, soluble, soluble(PEI clarified), soluble (after IDA column) and elution samples from theIDA column purification. The results demonstrated that all of the BotBprotein produced by the pHisBotB kan T7lac/pACYCGro/Bl21(DE3) cells wassoluble; the BotB protein was not precipitated by PEI treatment and wasquantitatively bound by the IDA column. Since a 1 liter fermentationharvest yielded a 75 gm cell pellet, this indicated that the yield ofsoluble affinity purified bot B protein from this fermentation was 12.5mg/liter. These results also demonstrated that additional purificationsteps are necessary to separate the chaperone proteins from the BotBprotein.

[0801] c) Fermentation of pHisBotBkan lacIq T7/pACYCGro/BL21(DE3) Cells

[0802] Induction was with 4 gms IPTG at 2 hr post initiation of theglucose feed. The OD₆₀₀ was 46 at time of induction, then 56, 63, 69 and71.5 at 1-4 hrs post induction. Viable colony counts decreased afterinduction (58, 3(5), 3, 0, 0 at 0-4 hr induction; bracketed numbersrepresent microcolonies; dilution 3 utilized 3 μL of dilution 2 cells).All (53/53) colonies scored at the time of induction retained the BotBplasmid (kan resistant) and the chaperone plasmid (chloramphenicolresistant) and no colonies at induction grew on IPTG+Kan plates (nomutations detected).

[0803] Total and soluble extracts were resolved on a 10% SDS-PAGE gelsand Western blotted and his-tagged proteins were detected utilizing theNiNTA-alkaline phosphatase conjugate. This analysis revealed that theGro chaperones were induced to high levels (observed by ponceau Sstaining), and a much higher expression of soluble Bot B protein(compared to expression in the pHisBotB kan T7lac/pACYCGro fermentation)was observed at all timepoints, including uninduced cells (some increasein BotB protein levels were observed after induction).

[0804] A small scale IDA purification of BotB protein from a 100 ml PEIclarified 15% w/v extract (15 gm cell pellet) yielded approximately 40mg protein, of which approximately 50% was BotB protein and 50% wasGroEL chaperone, as assessed by Coomassie staining of a 10% SDS-PAGEgel. The NiNTA alkaline phosphatase conjugate was utilized to detecthis-tagged proteins on a Western blot containing total, soluble, soluble(PEI clarified), soluble (after IDA column) and elution samples from theIDA column purification. The results demonstrated that a significantpercentage (i.e., −10-20%) of BotB protein was soluble, that thesolubilized protein was not precipitated by PEI treatment and wasquantitatively bound by the IDA column. Since a 10 liter fermentationyielded a 108 gm cell pellet, this indicated that the yield of solubleaffinity purified BotB protein from this fermentation was 144 mg/liter.

[0805] In a scale up experiment, 2 liters of a 20% w/v PEI clarifiedlysate of pHisBotB kan lacIq T7/pACYCGro/BL21(DE3) cells were purifiedon a large scale IDA column. The purification was performed induplicate. The total yield of BotB protein was 220 and 325 mgs proteinin the two experiments (assuming 1 mg/ml solution=2.0 OD₂₈₀/ml). Thisrepresents 0.7% or 1.0%, respectively, of the total soluble cellularprotein (assuming a PEI lystate having a concentration of 8 mgprotein/ml and that the eluted material comprises a 1:1 mixture of BotBand folding chaperone). The NiNTA alkaline phosphatase conjugate wasutilized to detect his-tagged proteins on a Western blot containingtotal, soluble, soluble (PEI clarified), soluble (after IDA column) andelution samples from the IDA column purification. These resultsdemonstrated that a significant percentage (i.e., ˜10-20%) of the BotBprotein was soluble, that the solubilized protein was not precipitatedby PEI treatment and was quantitatively bound by the IDA column. Since a1 liter fermentation harvest yielded a 108 gm cell pellet, thisindicated that the yield of soluble affinity purified BotB protein fromthe large scale purification was 60 mg or 89 mg/liter. These resultsalso demonstrated that further purification would be necessary to removethe contaminating chaperone protein.

[0806] The above results provide methodologies for the purification ofsoluble BotB protein from fermentation cultures, in a form contaminatedpredominantly with a single E. coli protein (the folding chaperoneutilized to enhance solubility). In the next example, methods areprovided for the removal of the contaminating chaperone protein.

EXAMPLE 40 Removal of Contaminating Folding Chaperone Protein FromPurified Recombinant C. botulinum Type B Protein

[0807] In this example size exclusion chromatography and ultrafiltrationwas used to purify recombinant BotB protein from the folding chaperonesand imidazole in IDA-purified material.

[0808] To enhance the solubility of the recombinant BotB protein duringscale-up, the protein was co-expressed with folding chaperones (see Ex.39). During the Ni-IDA purification step, the folding chaperonesco-eluted with the BotB protein in 800 mM imidazole; therefore, a secondpurification step was required to isolate the BotB free of foldingchaperones. Lane 3 of FIG. 35 contains proteins eluted from an IDAcolumn to which a lysate of pHisBotB kan lacIq T7/pACYCGro/BL21(DE3)cells had been applied; the proteins were resolved on a 4-15%polyacrylamide pre-cast gradient gel (Bio-Rad, Hercules, Calif.) rununder native conditions and then stained with Coomassie blue. In FIG.35, lanes 1 and 4 contain proteins present in peak 1 and peak 2 from aSephacryl S-100 column run as described below; lane 2 is blank.

[0809] As seen in lane 3 of FIG. 35, the IDA-purified sample consistsprimarily of the folding chaperones and the BotB protein. The fact thatthe chaperones and the Bot B antigen appear as two distinct bands undernative conditions suggested they were not complexed together andtherefore, it should be possible to separate them, using either agradient of imidazole concentrations or size exclusion methods.

[0810] In order to determine whether a gradient of imidazoleconcentrations could be used to separate the chaperone from the BotBprotein, a step gradient using imidazole at 200, 400, 600, and 800 mM in50 mM sodium phosphate, 0.5 M NaCl and 10% glycerol, pH 6.8 was appliedto an IDA column (containing proteins bound from a lysate of pHisBotBkan lacIq T7/pACYCGro/BL21(DE3) cells). By narrowing the range ofimidazole concentrations, it was hoped that the BotB and chaperoneproteins would differentially elute at different concentrations ofimidazole. Eluted proteins were monitored by absorbance at 280 nm andcollected either manually or with a fraction collector (BioRad). Proteinwas found to elute at 200 and 400 mM imidazole only.

[0811]FIG. 36 shows a Coomassie stained SDS-PAGE gel containing proteineluted during the imidazole step gradient. Lane 1 contains broad rangeMW markers (BioRad). Lane 2 contains BotB protein purified by IDAchromatography of an extract of pHisBotB/BL2l(DE3) pLysS cells grown inshaker flask culture (i.e., no co-expression of chaperones; Ex. 35).Lane 3 contains a 20% w/v PEI clarified lysate of pHisBotB kan lacIqT7/pACYCGro/BL21(DE3) cells (i.e., the lysate prior to purification byIDA chromatography). Lanes 4 and 5 contain protein which eluted at 200or 400 mM imidazole, respectively. Lane 6 is blank. Lanes 7 and 8contain ⅕ the load present in lanes 4 and 5.

[0812] As shown in FIG. 36, both the chaperone and the BotB proteineluted in 200 mM imidazole, and more chaperone elutes in 400 mMimidazole, however no concentration of imidazole tested permitted theelution of BotB protein alone. Consequently, no significant purificationwas achieved using imidazole at these concentrations.

[0813] Because of the considerable difference in molecular weightsbetween the folding chaperone, which is a multimer with a totalmolecular weight around 400 kD (as determined on a Shodex KB 804 sizingcolumn by HPLC), and the recombinant BotB protein (molecular weightaround 50 kD), size exclusion chromatography was next examined for theability to separate these proteins.

[0814] a) Size Exclusion Chromatography

[0815] A column containing Sephacryl S-100 HR(S-100) (Pharmacia) waspoured (2.5 cm×24 cm; ˜110 ml bed volume). The column was equilibratedin a buffer consisting of phosphate buffered saline (10 mM potassiumphosphate, 150 mM NaCl, pH 7.2) and 10% glycerol (Mallinkrodt).Typically, 5 ml of the IDA-purified BotB protein was filtered through a0.45μ syringe filter and applied to the column, and the equilibrationbuffer was pumped through the column at a flow rate of 1 ml/minute.Eluted proteins were monitored by absorbance at 280 nm and collectedeither manually or with a fraction collector. Appropriate tubes werepooled, if necessary, and the protein was quantitated by absorbance at280 nm and/or by BCA protein assay. The isolated peaks were thenanalyzed by native and/or SDS-PAGE to identify the protein and evaluatethe purity.

[0816] Because of its larger size, the folding chaperone eluted first,followed by the recombinant BotB protein. A smaller third peak wasobserved which failed to stain when analyzed by SDS-PAGE and thereforewas presumed to be imidazole.

[0817] SDS-PAGE analysis (12.5% polyacrylamide, reduced samples) wasused to evaluate the purity of the IDA-purified recombinant BotB proteinbefore and after S-100 purification. The results are shown in FIG. 33.

[0818] In FIG. 33, lane 1 contains broad range MW markers (BioRad). Lane5 contains IDA-purified BotB protein. Lane 6 contains IDA-purified BotBprotein following S-100 purification. Lane 7 is blank (lanes 2-4 werediscussed in Ex. 34 above).

[0819] The results shown in FIG. 33 show that the IDA-purified BotB issignificantly contaminated with the folding chaperone (molecular weightabout 60 kD under reducing conditions; lane 6). Following S-100purification, the amount of folding chaperone present in the BotB samplewas reduced dramatically (lane 7). Visual inspection of the Coomassiestained SDS-PAGE gel revealed that after S-100 purification, >90% of thetotal protein present was BotB.

[0820] The IDA-purified BotB and the S-100-purified BotB samples wereanalyzed by HPLC on a size exclusion column (Shodex KB 804); thisanalysis revealed that the BotB protein represented 64% of the totalprotein in the IDA-purified sample and that following S-100purification, the BotB protein represented >95% of the total protein inthe sample.

[0821] The IDA-purified BotB material was also applied to a ACA 44(SpectraPor, Houston, Tex.) column. The ACA 44 resin is equivalent tothe S-100 resin and chromatography using the ACA 44 resin was carriedout exactly as described above for the S-100 resin. The ACA 44 resin wasfound to separate the recombinant BotB protein from the foldingchaperone. The ACA 44-purified BotB sample was analyzed for endotoxinusing the LAL assay (Associates of Cape Cod) as describe in Example 24.Two aliqouts of the ACA 44-purified BotB preparation were analyzed andwere found to contain either 58 to 116 EU/mg recombinant protein or 94to 189 EU/mg recombinant protein.

[0822] These results demonstrate that size exclusion chromatography canbe used to purify the recombinant BotB protein from the foldingchaperone and imidazole in IDA-purified material

[0823] b) Ultrafiltration for the Separation of Recombinant BotB Proteinand Chaperones

[0824] Ultrafiltration was examined as an alternative method for theseparation recombinant BotB protein and folding chaperones inIDA-purified material. While in this example only mixtures of BotB andchaperones were separated by ultrafiltration, this technique is suitablefor use with recombinant BotA and BotE proteins as well provided thatthe wash buffers used are altered as necessary to take into accountdifferent requirements for solubility.

[0825] The recombinant BotB protein and folding chaperones wereseparated using a two-step sequential ultrafiltration method. The firstmembrane used had a nominal molecular weight cutoff (MWCO) ofapproximately 100 kD; this membrane retains the larger folding chaperonewhile allowing the smaller recombinant protein to pass through. Theaddition of several volumes of wash buffer may be required toefficiently wash the recombinant protein through the membrane. Thesecond step utilized a membrane with a nominal MWCO of approximately 10kD. During this step, the recombinant antigen was retained by themembrane and could be concentrated to the degree desired and theimidazole and excess wash buffer passed through the membrane.

[0826] Twenty-seven milliliters of an IDA-purified BotB preparation wasultrafiltered through a 47 mm YM 100 (100 kD MWCO) membrane (Amicon) ina 50 ml stirred cell (Amicon). The membrane was washed in dd H₂O priorto use as recommended by the manufacturer. Six volumes of 10% glycerolin PBS were washed through to remove most of the recombinant BotBprotein and this wash was collected in a separate vessel. The resultingBotB protein-rich filtrate was then concentrated 12-fold using a YM 10(10 kD MWCO) membrane (Amicon), to a final volume of 14 ml. The YM 100and YM 10 concentrates were analyzed along with the lysate startingmaterial by native PAGE using a 4-15% pre-cast gradient gel (BioRad).The results are shown in FIG. 37.

[0827] In FIG. 37, lane 1 contains IDA-purified BotB derived from ashaker flask culture (i e., no co-expression of chaperones; Ex. 35);lane 2 contains a 20% w/v PEI clarified lysate of pHisBotB kan lacIqT7/pACYCGro/BL21(DE3) cells; lane 3 shows the lysate of lane 3 after IDApurification; lane 4 contains the YM 10 concentrate and lane 5 containsthe YM 100 concentrate.

[0828] The results shown in FIG. 37 demonstrate that the recombinantBotB protein can be purified away from the folding chaperone byultrafiltration through a 100 kD MWCO membrane (lane 4), leaving thechaperone protein in the 100 kD concentrate (lane 5). Analysis of thesample in lane 5 also showed that very little of the BotB protein wasretained by the 100 kD MWCO membrane after 6 volumes of wash buffer hadbeen applied.

[0829] The BotB samples following IDA chromatography and followingultrafiltration through the YM 100 membrane were anlyzed by HPLC on asize exclusion column (Shodex KB 804); this analysis revealed that theBotB protein represented 64% of the total protein in the IDA-purifiedsample and that following ultrafiltration through the YM 100 membrane,the BotB protein represented >96% of the total protein in the sample.

[0830] The BotB protein purified by ultrafiltration through the YM 100membrane was examined for endotoxin using the LAL assay (Associates ofCape Cod) as describe in Example 24. Two aliqouts of the YM 100-purifiedBotB preparation were analyzed and were found to contain either 18 to 36EU/mg recombinant protein or 125 to 250 EU/mg recombinant protein.

[0831] The above results demonstrate that size exclusion chromatographyand ultrafiltration can be used to purify recombinant botulinal toxinproteins away from folding chaperones.

EXAMPLE 41 Cloning and Expression of the C Fragment of the C. botulinumSerotype E Toxin Gene

[0832] The C. botulinum type E neurotoxin gene has been cloned andsequenced from several different strains [Poulet et al. (1992) Biochem.Biophys. Res. Commun. 183:107 (strain Beluga); Whelan et al. (1992) Eur.J. Biochem. 204:657 (strain NCTC 11219); Fujii et al. (1990) Microbiol.Immunol. 34:1041 (partial sequence of strains Mashike, Iwani and Otaru)and Fujii et al. (1993) J. Gen. Microbiol. 139:79 (strain Mashike)]. Thenucleotide sequence of the type E toxin gene is available from the EMBLsequence data bank under accession numbers X62089 (strain Beluga) andX62683 (strain NCTC 11219). The nucleotide sequence of the coding region(strain Beluga) is listed in SEQ ID NO:49. The amino acid sequence ofthe C. botulinum type E neurotoxin derived from strain Belgua is listedin SEQ ID NO:50. The nucleotide sequence-of the coding region (strainNCTC 11219) is listed in SEQ ID NO:51. The amino acid sequence of the C.botulinum type E neurotoxin derived from strain NCTC 11219 is listed inSEQ ID NO:52.

[0833] The DNA sequence encoding the native C. botulinum serotype E Cfragment gene derived from the Beluga strain can be expressed as ahistidine-tagged protein using the pETHisb vector; the resulting codingregion is listed in SEQ ID NO:53 and the corresponding amino acidsequence is listed in SEQ ID NO:54. The DNA sequence encoding the Cfragment of the native C. botulinum serotype E gene derived from theNCTC 11219 strain can be expressed as a histidine-tagged fusion proteinusing the pETHisb vector; the resulting coding region is listed in SEQID NO:55 and the corresponding amino acid sequence is listed in SEQ IDNO:56. The C fragment region from any strain of C. botulinum serotype Ecan be amplified and expressed using the approach illustrated belowusing the C fragment derived from C. botulinum type E 2231strain (ATCC#17786).

[0834] The type E neurotoxin gene is synthesized as a single polypeptidechain which may be converted to a double-chain form (i.e., a heavy chainand a light chain) by cleavage with trypsin; unlike the type Aneurotoxin, the type E neurotoxin exists essentially only in thesingle-chain form. The 50 kD carboxy-terminal portion of the heavy chainis referred to as the C fragment or the H_(C) domain. Expression of theC fragment of C. botulinum type E toxin in heterologous hosts (e.g., E.coli) has not been previously reported.

[0835] The native C fragment of the C. botulinum serotype E toxin (BotE)gene was cloned and inserted into expression vectors to facilitateexpression of the recombinant BotE protein in E. coli. This exampleinvolved PCR amplification of the gene, cloning, and construction ofexpression vectors.

[0836] The BotE serotype gene was isolated using PCR as described forthe BotA serotype gene in Example 28. The C. botulinum type E strain wasobtained from the American Type Culture Collection (ATCC #17786; strain2231). The following primer pair was used in the PCR amplification:5′-CGCCATGGCTCTTTCTTCTTAT ACAGATGAT-3′ (5′ primer, engineered NcoI siteunderlined) (SEQ ID NO:57) and 5′-GCAAGCTTTTATTTTTCTTGCCATCCATG-3′ (3′primer, engineered HindIII site underlined, native gene terminationcodon italicized) (SEQ ID NO:58). The PCR product was inserted intopCRscript as described in Example 28. The resulting pCRscript BotE clonewas confirmed by restriction digestion, as well as, by obtaining thesequence of approximately 300 bases located at the 5′ end of the Cfragment coding region using standard DNA sequencing methods. Theresulting BotE sequence was identical to that of the published C.botulinum type E toxin sequence [Whelan et al (1992), supra].

[0837] The NheI(filled)/HindIII fragment from a pCRscript BotErecombinant was cloned into pETHisb vector as described for BotA Cfragment in Example 28. The resulting construct was termed pHisBotE.pHisBotE expresses the BotE gene under the control of the T7 lacpromoter and the resulting protein contains an N-terminal 10×His-tagaffinity tag.

[0838] The pHisBotE expression construct was transformed into BL21(DE3)pLysS competent cells and 1 liter cultures were grown, induced andhis-tagged proteins were purified utilizing a NiNTA resin (eluted in lowpH elution buffer) as described in Example 28. Total, soluble andpurified proteins were resolved by SDS-PAGE and detected by Coomassiestaining. The results are shown in FIG. 38.

[0839] In FIG. 38, lane 1 contains broad range MW markers (BioRad); lane2 contains a total protein extract; lane 3 contains a soluble proteinextract; lane 4 contains proteins present in the flow through from theNiNTA column (this sample was not diluted prior to loading and thereforerepresents a load 5× that of the load applied for the total and solubleextracts in lanes 2 and 3); lane 5 contains proteins eluted from theNiNTA column; lane 6 contains protein eluted from a NiNTA column whichhad been stored at −20° C. for 1 year.

[0840] The pHisBotE protein was expressed at moderate levels (7mg/liter) as a totally soluble protein. The purified protein migrated asa single band of the predicted MW.

[0841] Western blot hybridization utilizing a chicken anti-C. botulinumserotype E toxoid primary antibody (generated by immunization of hens asdescribed in Example 3 using C. botulinum serotype E toxoid) was alsoperformed on the total, soluble and purified BotE proteins. Samples ofBotA and BotB C fragments were also included on the gels to facilitateMW and immunogenicity comparisons. Strong immunoreactivity was detectedusing the anti-C. botulinum type E toxoid antibody only with the BotEprotein.

[0842] These results demonstrate that the native BotE gene sequences canbe expressed as a soluble his-tagged protein in E. coli and purified bymetal-chelation affinity chromatography.

EXAMPLE 42 Generation of Neutralizing Antibodies Using the RecombinantpHisBotE Protein

[0843] The ability of the purified pHisBotE protein to generateneutralizing antibodies was examined. Nine BALBc mice were immunizedwith BotE protein (purified as described in Ex. 41) using Gerbu GMDPadjuvant (CC Biotech). The low pH elution was mixed with Gerbu adjuvantand used to immunize mice. Each mouse received a subcutaneous injectionof 100 μl antigen/adjuvant mix (35 μg antigen+1 μg adjuvant) on day 0.Mice were subcutaneously boosted as above on day 14 and bled on day 28.Mice were subsequently boosted and bled on day 70.

[0844] Anti-C. botulinum serotype E toxoid titers were determined in day28 serum from individual mice from each group using the ELISA protocoloutlined in Example 29 with the exception that the plates were coatedwith C. botulinum serotype E toxoid, and the primary antibody was achicken anti-C. botulinum serotype E toxoid. Seroconversion [relative tocontrol mice immunized with the p6×HisBotA antigen (Ex. 29)] wasobserved with all 9 mice immunized with the purified pHisBotE protein.

[0845] The ability of the anti-BotE antibodies to neutralize native C.botulinum type E toxin was tested in a mouse-C. botulinum neutralizationmodel using pooled mouse serum (see Ex. 23b). The LD₅₀ of purified C.botulinum type E toxin complex (Dr. Eric Johnson, University ofWisconsin, Madison) was determined by a intraperitoneal (IP) method[Schantz and Kautler (1978), supra] using 18-22 g female ICR mice. Theamount of neutralizing antibodies present in the serum of the immunizedmice was determined using serum antibody titrations. The various serumdilutions (0.01 ml) were mixed with 5 LD₅₀ units of C. botulinum type Etoxin and the mixtures were injected IP into mice. The neutralizationswere performed in duplicate. The mice were then observed for signs ofbotulism for 4 days. Undiluted serum from day 28 did not protect, whileundiluted, {fraction (1/10)} diluted and {fraction (1/100)} diluted day70 serum protected (1005 of animals) while {fraction (1/1000)} dilutedday 70 serum did not. This corresponds to a neutralization titer of50-500 IU/ml.

[0846] These results demonstrate that seroconversion occurred andneutralizing antibodies were induced when the recombinant BotE proteinwas utilized as the immunogen.

EXAMPLE 43 Construction of Vectors to Facilitate Expression ofHis-Tagged BotE Protein in Fermentation Cultures

[0847] A number of expression vectors were constructed to facilitate theexpression of recombinant BotE protein in large scale fermentationculture. These constructs varied as to the strength of the promoterutilized (T7 or T7lac) and the presence of repressor elements (lacIq) onthe plasmid. The resulting constructs varied in the level of expressionachieved and in plasmid stability which facilitated the selection of aoptimal expression system for fermentation scaleup. This exampleinvolved a) construction of BotE expression vectors and b) determinationof expression levels in small scale cultures.

[0848] a) Construction of BotE Expression Vectors

[0849] The BotE expression vectors created for fermentation culture wereengineered to utilize the kanamycin rather than the ampicillinresistance gene, and contained either the T7 or T7lac promoter, with orwithout the lacIq gene for the reasons outlined in Example 30.

[0850] In all cases, the protein expressed by the various expressionvectors is the pHisBotE protein described in Example 41, with the onlydifferences between clones being the alteration of various regulatoryelements. Using the designations outlined below, the pHisBotE clone (Ex.41) is equivalent to pHisBotE amp T7lac.

[0851] i) Construction of pHisBotE kan lacIq T7lac

[0852] pHisBotE kan lacIq T7lac was constructed by inserting theXbaI/HindIII fragment of pHisBotE which contains the BotE gene sequencesinto XbaI/HindIII-cleaved pET24a vector. Proper construction wasconfirmed by restriction digestion.

[0853] ii) Construction of pHisBotE kan T7

[0854] pHisBotE kan T7 was constructed by ligating the BotE-containingXbaI/SapI fragment of pHisBotE kan lacIqT7lac to the T7promoter-containing XbaI/SapI fragment of pET23a. Proper constructionwas confirmed by restriction digestion.

[0855] iii) Construction of pHisBotE kan lacIqT7

[0856] pHisBotE kan lacIqT7 was constructed by inserting theBglII/HindIII fragment from pHisBotE kan T7 which contains the BotE genesequences into BglII/HindIII-cleaved pET24 vector. Proper constructionwas confirmed by restriction digestion.

[0857] b) Determination of BotE Expression Levels in Small ScaleCultures

[0858] The three BotE kan expression vectors described above weretransformed into Bl21(DE3) competent cells and 50 ml (2×YT+40 μg/ml kan)cultures were grown and induced with ITPG as described in Example 28.Total and soluble protein extracts from before and after induction madeas described in Example 28. The total and soluble extracts were resolvedon a 12.5% SDS-PAGE gel, and his-tagged proteins were detected on aWestern blot utilizing the NiNTA-alkaline phosphatase conjugate asdescribed in Example 31(c)(iii). The results showed that all three BotEcell lines expressed his-tagged proteins of the predicted MW for theBotE protein upon induction. The results also demonstrated that the twoconstructs that contained the T7 promoter expressed the BotE proteinbefore induction, while the T7lac promoter construct did not. Uponinduction, the T7 promoter-containing constructs induced to higherlevels than the T71α-containing construct, with the pHisBotE kanlacIqT7/Bl21(DE3) cells accumulating the maximal levels of BotE protein.

EXAMPLE 44 Expression and Purification of pHisBotE from FermentationCultures

[0859] Based on the small scale inductions performed in Example 43, thepHisBotE kan lacIq T7/Bl21(DE3) strain was selected for fermentationscaleup. This example involved the fermentation and purification ofrecombinant BotE C fragment protein.

[0860] A fermentation with the pHisBotE kan lacIq T7/Bl21(DE3) strainwas performed as described in Example 31. The fermentation culture wasinduced 2 hrs post start of the glucose feed with 4 gm IPTG (finalconcentration=1.6 mM). The OD₆₀₀ was 42 at time of induction, then 46.5,48, 53 and 54 at 1-4 hrs post induction. Viable colony counts decreasedfrom 0-4 hr induction [131, 4 (28), 7 (3), 7, 8; dilution 3 utilized 6μl of dilution 2 cells; bracketed colonies are microcolonies]. All(32/32) colonies scored at the time of induction retained the BotEplasmid (kan resistant) and no colonies at induction grew on IPTG+Kanplates (no mutations detected). These results were indicative of strongpromoter induction, since colony viability reduced after induction, andthe culture stopped growing during fermentation (stopped at 54 OD₆₀₀ml).

[0861] Total and soluble extracts were resolved on a 12.5% SDS-PAGE geland total protein was detected by staining with Coomassie blue. Theresults are shown in FIG. 39.

[0862] In FIG. 39, lane 1 contains total protein from a pHisBotA kan T7lac/Bl21(DE3) pLysS fermentation (Ex. 24). Lanes 2-9 contain extractsprepared from the above pHisBotE kan lacIq T7/Bl21(DE3) fermentation;lanes 2-4 contain total protein extracts prepared at 0, 1 and 2 hourspost-induction, respectively. Lane 5 contains a soluble protein extractprepared at 2 hours post-induction. Lanes 6 and 7 contain total andsoluble extracts prepared at 3 hours post-induction, respectively. Lanes8 and 9 contain total and soluble extracts prepared at 4 hourspost-induction, respectively. Lane 10 contains broad range MW markers(BioRad).

[0863] The results shown in FIG. 39 demonstrate that moderate levelinduction of totally soluble Bot E protein was observed, increasing from1 to 4 hrs post induction (no expression was detected in uninducedcells). From a 2 liter fermentation harvest a 155 gm (wet wt) cellpellet was obtained and used to make a PEI-clarified lysate (1 liter inCRB, pH 6.8). The lysate was applied to a large scale IDA column and 200mg of BotE protein, which was found to be greater than 95% pure (asjudged by visual inspection of a Coomassie stained SDS-PAGE gel), wasrecovered. This represents 2.5% of the total soluble cellular protein(assuming a PEI lysate having a concentration of 8 mg protein/ml) andcorresponds to a yield of 100 mg BotE protein/liter of fermentationculture.

[0864] The above results demonstrate that high levels of the recombinantBotE protein can be expressed and purified from fermentation cultures.

EXAMPLE 45 Removal of Imidazole from Purified Recombinant BotE ProteinPreparations

[0865] The expression of recombinant BotE protein, unlike the BotA andBotB proteins, did not require the presence of folding chaperones tomaintain solubility during scale-up. A size exclusion chromatographystep was included however to remove the imidazole from the sample andexchange the IDA elution buffer for one consistent with the BotAantigen.

[0866] A Sephacryl S-100 HR(S-100; Pharmacia) column was poured (2.5cm×24 cm; bed volume˜110 ml). Under these conditions, the BotE proteinshould be retained by the beads to a lesser degree than the smallerimidazole, therefore the BotE protein should elute from the columnbefore the imidazole. The column was equilibrated in a buffer consistingof 50 mM sodium phosphate, 0.5 M NaCl, and 10% glycerol (all reagentsfrom Mallinkrodt). Five milliliters of the IDA-purified BotE protein(Ex. 44) was filtered through a 0.45μ syringe filter and applied to theS-100 column, and equilibration buffer was pumped through the column ata flow rate of 1 ml/minute. Eluted proteins were monitored by absorbanceat 280 nm, and collected either manually or with a fraction collector.Appropriate tubes were pooled if necessary, and the protein wasquantitated by absorbance at 280 nm and/or BCA protein assay. Theisolated peaks were then analyzed by native and/or SDS-PAGE to identifythe protein(s) and evaluate the purity.

[0867]FIG. 40 provides a representative chromatogram showing thepurification of IDA-purified BotE on the S-100 column. Even thoughfolding chaperones were not over-expressed with this antigen, a smallamount of protein eluted at a time consistent with the foldingchaperones expressed with BotA and BotB proteins (Gro) (see the firstpeak). The second peak in the chromatogram contained the BotE protein,and the third peak was presumably imidazole. This presumed imidazolepeak was isolated in comparable levels in IDA-purified BotA and BotBprotein preparations as well.

[0868] These results demonstrate that size exclusion chromatography canbe used to remove imidazole and traces of contaminating high molecularweight proteins from IDA-purified BotE protein preparations.

[0869] The S-100-purified BotE protein was tested for endotoxincontamination using the LAL assay as described in Example 24. Thispreparation was found to contain 64 to 128 EU/mg recombinant protein andis therefore substantially free of endotoxin.

[0870] The S-100 purified BotE was mixed with purified preparations ofBotA and BotB proteins and used to immunize mice; 5 μg of each Botprotein was used per immunization and alum was included as an adjuvant.After two immunizations with this trivalent vaccine, the immunized micewere challanged with C. botulinum toxin. The immunized mice containedneutralizing antibodies sufficient to neutralize between 100,000 to1,000,000 LD₅₀ of either toxin A or toxin B and between 1,000 to 10,000LD₅₀ of toxin E. The titer of neutralizing antibodies directed againsttoxin E would be expected to increase following subsequent boosts withthe vaccine. These results demonstrate that a trivalent vaccinecontaining recombinant BotA, BotB and BotE proteins provokesneutralizing antibodies.

EXAMPLE 46 Expression of the C Fragment of the C. botulinum Serotype CToxin Gene and Generation of Neutralizing Antibodies

[0871] The C. botulinum type Cl neurotoxin gene has been cloned andsequenced [Kimura et al (1990) Biochem. Biophys. Res. Comm. 171:1304].The nucleotide sequence of the toxin gene derived from the C. botulinumtype C strain C-Stockholm is available from the EMBL/GenBank sequencedata banks under the accession number D90210; the nucleotide sequence ofthe coding region is listed in SEQ ID NO:59. The amino acid sequence ofthe C. botulinum type C1 neurotoxin derived from this strain is listedin SEQ ID NO:60.

[0872] The DNA sequence encoding the native C. botulinum serotype C₁-Cfragment gene derived from the C-Stockholm strain can be expressed usingthe pETHisb vector; the resulting coding region is listed in SEQ IDNO:61 and the corresponding amino acid sequence is listed in SEQ IDNO:62. The C fragment region from any strain of C. botulinum serotype Ccan be amplified and expressed using the approach illustrated belowusing the C fragment derived from C. botulinum type C C-Stockholmstrain. Expression of the C fragment of C. botulinum type Cl toxin inheterologous hosts (e.g., E. coli) has not been previously reported.

[0873] The C fragment of the C. botulinum serotype C1 (BotC1) toxin geneis cloned using the protocols and conditions described in Example 28 forthe isolation of the native BotA gene. A number of C. botulinum serotypeC strains (expressing either or both C1 and C2 toxin) are available fromthe ATCC [eg., 2220 (ATCC 17782), 2239 (ATCC 17783), 2223 (ATCC 17784; atype C-β strain; C-β strains produce C2 toxin), 662 (ATCC 17849; a typeC-α strain; C-α strains produce mainly C1 toxin and a small amount of C2toxin), 2021 (ATCC 17850; a type C-α strain) and VPI 3803 (ATCC 25766)].Alternatively, other type C strains may be employed for the isolation ofsequences encoding the C fragment of C. botulinum serotype C toxin.

[0874] The following primer pair is used to amplify the BotC gene:5′-CGCCATGGC TTTATTAAAAGATATAATTAATG-3′ [5′ primer, engineered NcoI siteunderlined (SEQ ID NO:63)] and 5′-GCAAGCTTTTATTCACTTACAGGTAC AAAACC-3′[3′ primer, engineered HindIII site underlined, native gene terminationcodon italicized (SEQ ID NO:64)]. Following PCR amplification, the PCRproduct is inserted into the pCRscript vector and then the 1.5 kbfragment is cloned into pETHisb vector as described for BotA C fragmentgene in Example 28. The resulting construct is termed pHisBotC. Properconstruction is confirmed by DNA sequencing of the BotC sequencescontained within pHisBotC.

[0875] pHisBotC expresses the BotC gene sequences under thetranscriptional control of the T7 lac promoter and the resulting proteincontains an N-terminal 10×His-tag affinity tag. The pHisBotC expressionconstruct is transformed into BL21(DE3) pLysS competent cells and 1liter cultures are grown, induced and his-tagged proteins are purifiedutilizing a NiNTA resin (eluted in 250 mM imidazole, 20% glycerol) asdescribed in Example 28. Total, soluble and purified proteins areresolved by SDS-PAGE and detected by Coomassie staining and Western blothybridization utilizing a Ni-NTA-alkaline phosphatase conjugate (Qiagen)which recognizes his-tagged proteins as described in Example 31(c)(iii). This analysis permits the determination of expression levelsof the pHisBotC protein (i.e., number of mg/liter expressed as a solubleprotein). The purified BotC protein will migrate as a single band of thepredicted MW (i.e., ˜50 kD).

[0876] The level of expression of the pHisBotC protein may be modified(increased) by substitution of the T7 promoter for the T7lac promoter,or by inclusion of the lacIq gene on the expression plasmid, and plasmidexpressed in BL21(DE3) cell lines in fermentation cultures as describedin Example 30. If only very low levels (i.e., less than 0.5%) of solublepHisBotC protein are expressed using the above expression systems, thepHisBotC construct may be co-expressed with pACYCGro construct asdescribed in Example 32. In this case, the recombinant BotC protein mayco-purify with the folding chaperones. The contaminating chaperones maybe removed as described in Example 34. Preparations of purified pHisBotCprotein are tested for endotoxin contamination using the LAL assay asdescribed in Example 24.

[0877] The purifed pHisBotC protein is used to generate neutralizingantibodies. BALBc mice are immunized with the BotC protein using GerbuGMDP adjuvant (CC Biotech) as described in Example 36. The ability ofthe anti-BotC antibodies to neutralize native C. botulinum type C toxinis demonstrated using the mouse-C. botulinum neutralization modeldescribed in Example 36.

EXAMPLE 47 Expression of the C Fragment of the C. botulinum Serotype DToxin Gene and Generation of Neutralizing Antibodies

[0878] The C. botulinum type D neurotoxin gene has been cloned andsequenced [Sunagawa et al. (1992) J. Vet. Med. Sci. 54:905 and Binz etal. (1990) Nucleic Acids Res. 18:5556]. The nucleotide sequence of thetoxin gene derived from the CB16 strain is available from theEMBL/GenBank sequence data banks under the accession number S49407; thenucleotide sequence of the coding region is listed in SEQ ID NO:65. Theamino acid sequence of the C. botulinum type D neurotoxin derived fromthe CB16 strain is listed in SEQ ID NO:66.

[0879] The DNA sequence encoding the native C. botulinum serotype D Cfragment gene derived from a BotD expressing strain can be expressedusing the pETHisb vector; the resulting coding region is listed in SEQID NO:67 and the corresponding amino acid sequence is listed in SEQ IDNO:68. The C fragment region from any strain of C. botulinum serotype Dcan be amplified and expressed using the approach illustrated belowusing the C fragment derived from C. botulinum type D CB16 strain.Expression of the C fragment of C. botulinum type D toxin inheterologous hosts (e.g. E. coli) has not been previously reported.

[0880] The C fragment of the C. botulinum serotype D (BotD) toxin geneis cloned using the protocols and conditions described in Example 28 forthe isolation of the native BotA gene. A number of C. botulinum type Dstrains are available from the ATCC [e.g., ATCC 9633, 2023 (ATCC 17851),and VPI 5995 (ATCC 27517)].

[0881] The following primer pair is used to amplify the BotD gene:5′-CGCCATGGC MATTAAAAGATATAATTAATG-3′ [5′ primer, engineered NcoI siteunderlined (SEQ ID NO:63)] and 5′-GCAAGCTTTTACTCTACCCATCCTGGATCCCT-3′[3′ primer, engineered HindIII site underlined, native gene terminationcodon italicized (SEQ ID NO:69)]. Following PCR amplification, the PCRproduct is inserted into the pCRscript vector and then the 1.5 kbfragment is cloned into pETHisb vector as described for BotA C fragmentgene in Example 28. The resulting construct is termed pHisBotD. pHisBotDexpresses the BotD gene sequences under the transcriptional control ofthe T7 lac promoter and the resulting protein contains an N-terminal10×His-tag affinity tag. The pHisBotD expression construct istransformed into BL21(DE3) pLysS competent cells and 1 liter culturesare grown, induced and his-tagged proteins are purified utilizing aNiNTA resin as described in Example 28. Total, soluble and purifiedproteins are resolved by SDS-PAGE and detected by Coomassie staining andWestern blot hybridization utilizing a Ni-NTA-alkaline phosphataseconjugate (Qiagen) which recognizes his-tagged proteins as described inExample 31 (c)(iii). This analysis permits the determination ofexpression levels of the pHisBotD protein (i.e., number of mg/literexpressed as a soluble protein). The purified BotD protein will migrateas a single band of the predicted MW (i.e., ˜50kD).

[0882] The level of expression of the pHisBotD protein may be modified(increased) by substitution of the T7 promoter for the T7lac promoter,or by inclusion of the lacIq gene on the expression plasmid, and plasmidexpressed in BL21(DE3) cell lines in fermentation cultures as describedin Example 30. If only very low levels (i.e., less than about 0.5%) ofsoluble pHisBotD protein are expressed using the above expressionsystems, the pHisBotD construct may be co-expressed with pACYCGroconstruct as described in Example 32. In this case, the recombinant BotDprotein may co-purify with the folding chaperones. The contaminatingchaperones may be removed as described in Example 34. Preparations ofpurified pHisBotD protein are tested for endotoxin contamination usingthe LAL assay as described in Example 24.

[0883] The purifed pHisBotD protein is used to generate neutralizingantibodies. BALBc mice are immunized with the BotD protein using GerbuGMDP adjuvant (CC Biotech) as described in Example 36. The ability ofthe anti-BotD antibodies to neutralize native C. botulinum type D toxinis demonstrated using the mouse-C. botulinum neutralization modeldescribed in Example 36.

EXAMPLE 48 Expression of the C Fragment of the C. botulinum Serotype FToxin Gene and Generation of Neutralizing Antibodies

[0884] The C. botulinum type F neurotoxin gene has been cloned andsequenced [East et al. (1992) FEMS Microbiol. Lett. 96:225]. Thenucleotide sequence of the toxin gene derived from the 202F strain (ATCC23387) is available from the EMBL/GenBank sequence data banks under theaccession number M92906; the nucleotide sequence of the coding region islisted in SEQ ID NO:70. The amino acid sequence of the C. botulinum typeF neurotoxin derived from the 202F strain is listed in SEQ ID NO:71.

[0885] The DNA sequence encoding the native C. botulinum serotype F Cfragment gene derived from the 202F strain can be expressed using thepETHisb vector; the resulting coding region is listed in SEQ ID NO:72and the corresponding amino acid sequence is listed in SEQ ID NO:73. TheC fragment region from any strain of C. botulinum serotype F can beamplified and expressed using the approach illustrated below using the Cfragment derived from C. botulinum type F 202F strain. Expression of theC fragment of C. botulinum type F toxin in heterologous hosts (e.g., E.coli) has not been previously reported.

[0886] The C fragment of the C. botulinum serotype F (BotF) toxin geneis cloned using the protocols and conditions described in Example 28 forthe isolation of the native BotA gene. The C. botulinum type F 202Fstrain is obtained from the American Type Culture Collection (ATCC23387). Alternatively, sequences encoding the BotF toxin may be isolatedfrom any BotF expressing strain [e.g., VPI 4404 (ATCC 25764), VPI 2382(ATCC 27321) and Langeland (ATCC 35415)]. The following primer pair isused to amplify the BotF gene: 5′-CGCCATGGC TATTCTAATTATATATTTTAATAG-3′[5′ primer, engineered NcoI site underlined (SEQ ID NO:74)] and5′-GCAAGCTTTCATTCTTTCCATCCATTCTC-3′ [3′ primer, engineered HindIII siteunderlined, native gene termination codon italicized (SEQ ID NO:75)].Following PCR amplification, the PCR product is inserted into thepCRscript vector and then the 1.5 kb fragment is cloned into pETHisbvector as described for BotA C fragment gene in Example 28. Theresulting construct is termed pHisBotF. pHisBotF expresses the BotF genesequences under the transcriptional control of the T7 lac promoter andthe resulting protein contains an N-terminal 10×His-tag affinity tag.The pHisBotF expression construct is transformed into BL21(DE3) pLysScompetent cells and 1 liter cultures are grown, induced and his-taggedproteins are purified utilizing a NiNTA resin as described in Example28. Total, soluble and purified proteins are resolved by SDS-PAGE anddetected by Coomassie staining and Western blot hybridization utilizinga Ni-NTA-alkaline phosphatase conjugate (Qiagen) which recognizeshis-tagged proteins as described in Example 31 (c)(iii). This analysispermits the determination of expression levels of the pHisBotF protein(i.e., number of mg/liter expressed as a soluble protein). The purifiedBotF protein will migrate as a single band of the predicted MW (i.e.,˜50 kD).

[0887] The level of expression of the pHisBotF protein may be modified(increased) by substitution of the T7 promoter for the T7lac promoter,or by inclusion of the lacIq gene on the expression plasmid, and plasmidexpressed in BL21(DE3) cell lines in fermentation cultures as describedin Example 30. If only very low levels (i.e., less than about 0.5%) ofsoluble pHisBotF protein are expressed using the above expressionsystems. the pHisBotF construct may be co-expressed with pACYCGroconstruct as described in Example 32. In this case, the recombinant BotFprotein may co-purify with the folding chaperones. The contaminatingchaperones may be removed as described in Example 34. Preparations ofpurified pHisBotF protein are tested for endotoxin contamination usingthe LAL assay as described in Example 24.

[0888] The purifed pHisBotF protein is used to generate neutralizingantibodies. BALBc mice are immunized with the BotF protein using GerbuGMDP adjuvant (CC Biotech) as described in Example 36. The ability ofthe anti-BotF antibodies to neutralize native C. botulinum type F toxinis demonstrated using the mouse-C. botulinum neutralization modeldescribed in Example 36.

EXAMPLE 49 Expression of the C Fragment of the C. botulinum Serotype GToxin Gene and Generation of Neutralizing Antibodies

[0889] The C. botulinum type G neurotoxin gene has been cloned andsequenced [Campbell et al. (1993) Biochimica et Biophysica Acta 1216:487and Binz et al. (1990) Nucleic Acids Res. 18:5556]. The nucleotidesequence of the toxin gene derived from the 113/30 strain (NCFB 3012) isavailable from the EMBL/GenBank sequence data banks under the accessionnumber X74162; the nucleotide sequence of the coding region is listed inSEQ ID NO:76. The amino acid sequence of the C. botulinum type Gneurotoxin derived from this strain is listed in SEQ ID NO:77.

[0890] The DNA sequence encoding the native C. botulinum serotype G Cfragment gene derived from the 113/30 strain can be expressed using thepETHisb vector; the resulting coding region is listed in SEQ ID NO:78and the corresponding amino acid sequence is listed in SEQ ID NO:79. TheC fragment region from any strain of C. botulinum serotype G can beamplified and expressed using the approach illustrated below using the Cfragment derived from C. botulinum type G 113/30 strain. Expression ofthe C fragment of C. botulinum type G toxin in heterologous hosts (e.g.,E. coli) has not been previously reported.

[0891] The C fragment of the C. botulinum serotype G (BotG) toxin geneis cloned using the protocols and conditions described in Example 28 forthe isolation of the native BotA gene. The C. botulinum type G 113/30strain is obtained from the NCFB. The following primer pair is used toamplify the BotG gene: 5′-CGCCATGGCTGAC ACAATTTTAATACA AGT-3′ [5′primer, engineered NcoI site underlined (SEQ ID NO:80)] and5′-GCCTCGAGTTATTCTGTCCATCCTTCATCCAC-3′ [3′ primer, engineered XhoI siteunderlined, native gene termination codon italicized (SEQ ID NO:81)].Following PCR amplification, the PCR product is inserted into thepCRscript vector and then the 1.5 kb fragment is cloned into pETHisbvector as described for BotA C fragment gene in Example 28 with theexception that the sequences encoding BotG are excised from thepCRscript vector by digestion with NcoI and XhoI and the NcoI site isblunted (the BotG sequences contain an internal HindIII site). ThisNcoI(filled)/XhoI fragment is then ligated to the pETHisb vector whichhas been digested with NheI and Salt and the NheI site is blunted. Theresulting construct is termed pHisBotG. pHisBotG expresses the BotG genesequences under the transcriptional control of the T7 lac promoter andthe resulting protein contains an N-terminal 10×His-tag affinity tag.The pHisBotG expression construct is transformed into BL21(DE3) pLysScompetent cells and 1 liter cultures are grown, induced and his-taggedproteins are purified utilizing a NiNTA resin as described in Example28. Total, soluble and purified proteins are resolved by SD S-PAGE anddetected by Coomassie staining and Western blot hybridization utilizinga Ni-NTA-alkaline phosphatase conjugate (Qiagen) which recognizeshis-tagged proteins as described in Example 31 (c)(iii). This analysispermits the determination of expression levels of the pHisBotG protein(i.e., number of mg/liter expressed as a soluble protein). The purifiedBotG protein will migrate as a single band of the predicted MW (i.e.,−50kD).

[0892] The level of expression of the pHisBotG protein may be modified(increased) by substitution of the T7 promoter for the T7ac promoter, orby inclusion of the lacIq gene on the expression plasmid, and plasmidexpressed in BL21(DE3) cell lines in fermentation cultures as describedin Example 30. If only very low levels (i.e., less than about 0.5%) ofsoluble pHisBotG protein are expressed using the above expressionsystems, the pHisBotG construct may be co-expressed with pACYCGroconstruct as described in Example 32. In this case, the recombinant BotGprotein may co-purify with the folding chaperones. The contaminatingchaperones may be removed as described in Example 34. Preparations ofpurified pHisBotG protein are tested for endotoxin contamination usingthe LAL assay as described in Example 24.

[0893] The purifed pHisBotG protein is used to generate neutralizingantibodies. BALBc mice are immunized with the BotG protein using GerbuGMDP adjuvant (CC Biotech) as described in Example 36. The ability ofthe anti-BotG antibodies to neutralize native C. botulinum type G toxinis demonstrated using the mouse-C. botulinum neutralization modeldescribed in Example 36.

EXAMPLE 50 Expression of Recombinant Botulinal Toxin Proteins inEucaryotic Host Cells

[0894] Recombinant botulinal C fragment proteins may be expressed ineucaryotic host cells, such as yeast and insect cells.

[0895] a) Expression in Yeast

[0896] Botulinal C fragments derived from serotypes A, B, C, D, E, F andG may be expressed in yeast cells using a variety of commerciallyavailable vectors. For example, the pPIC3K and pPIC9K expression vectors(Invitrogen) may be employed for expression in the methylotrophic yeast,Pichia pastoris. When the pPIC3K vector is employed, expression of thebotulinal C fragment protein will be intracellular. When the pPIC3Kvector is employed, the botulinal C fragment protein will be secreted(the alpha factor secretion signal is provided on the pPIC9K vector).

[0897] DNA sequences encoding the desired C fragment is inserted intothese vectors using techniques known to the art. Briefly, the desiredbotulinal expression cassette (including sequences encoding the his-tag;described in the preceding examples) is amplified using the PCR inconjunction with primers that incorporate unique restriction sites atthe termini of the amplified fragment. Suitable restriction enzyme sitesinclude SnaBI, EcoRI, AvrII and NotI. When the botulinal C fragment isto be expressed using the pPIC3K vector, the initiator methionine (ATG)is provided by the desired Bot gene sequence and a Kozak consensussequence is engineered upstream of the ATG (e.g., ACCATGG).

[0898] The amplified restriction fragment containing the botulinal Cfragment gene is then cloned into the desired expression vector.Recombinant clones are integrated into the Pichia pastoris genome andrecombinant protein expression is induced using methanol following themanufacturer's instructions (Invitrogen Pichia expression kit manual).

[0899]C. botulinum genes are A/T rich and contain multiple sequencesthat are similar to yeast transcriptional termination signals (e.g.,TTTTTATA). If premature transcription termination is observed when thebotulinal C fragment genes are expressed in yeast, the transcriptiontermination signals present in the C fragment genes can be removed byeither site directed mutagenesis (utilizing the pALTER system; Promega)or by construction of synthetic genes utilizing overlapping syntheticprimers.

[0900] The botulinal C fragment genes may be expressed in other yeastcells using other commercially available vectors [e.g., using the pYES2vector (Invitrogen) and S. cerevisiae cells (Invitrogen)].

[0901] b) Expression in Insect Cells

[0902] Botulinal C fragments derived from serotypes A, B, C, D, E, F andG may be expressed in insect cells using a variety of commerciallyavailable vectors. For example, the pBlueBac4 transfer vector(invitrogen) may be employed for expression in Spodoptera frugiperda(Sf9) insect cells (baculovirus expression system) (equivalentbaculovirus vectors and host cells are avaialble from other vendors,e.g., Pharmingen, San Diego, Calif.). Botulinal C fragments contained onNcoI/HindIII fragments contained within the pHisBotA-G expressionconstructs (described in the preceding examples) are cloned into thepBlueBac4 vector (digested with NcoI and HindIII); the NcoI site presenton the C fragment constructs overlaps with the start codon of the fusionproteins. In the case of botulinal C fragment clones that containinternal HindIII sites (e.g., using the BotG sequences described in Ex.49), the C fragment gene is contained within a NcoI/XhoI fragment on thepHisBot construct. This NcoI/XhoI fragment is excised from pHisBot andinserted into pBlueBac4 digested with NcoI and SalI. Recombinantbaculoviruses are made and the desired recombinant C fragment isexpressed in Sf9 cells using the protocols provided by the manufacturer(Invitrogen MaxBac manual). The resulting constructs will express thepHisBot protein intracellularly (including the N-terminal his-tag) underthe control of the polyhedrin promoter. For extracellular secretion ofbotulinal C fragment proteins, the C fragment sequences from the pHisBotconstructs are cloned into the pMelBacB vector (Invitrogen) as describedabove for the pBlueBac4 vector. When the pMelBacB vector is employed,the his-tagged botulinal C fragment proteins are secreted (utilizing avector-encoded honeybee melittin secretion signal) and contain a nineamino acid extension at the N-terminus.

[0903] His-tagged botulinal C fragments expressed in yeast or insectcells are purified using metal chelation columns as described in thepreceding examples.

[0904] From the above it is clear that the present invention providescompositions and methods for the preparation of effective multivalentvaccines against C. botulinum neurotoxin. It is also contemplated thatthe recombinant botulinal proteins be used for the production ofantitoxins. All publications and patents mentioned in the abovespecification are herein incorporated by reference. Variousmodifications and variations of the described method and system of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention.

1 82 24 base pairs nucleic acid single linear DNA (genomic) 1 GGAAATTTAGCTGCAGCATC TGAC 24 24 base pairs nucleic acid single linear DNA(genomic) 2 TCTAGCAAAT TCGCTTGTGT TGAA 24 20 base pairs nucleic acidsingle linear DNA (genomic) 3 CTCGCATATA GCATTAGACC 20 19 base pairsnucleic acid single linear DNA (genomic) 4 CTATCTAGGC CTAAAGTAT 19 8133base pairs nucleic acid single linear DNA (genomic) CDS 1..8130 5 ATGTCT TTA ATA TCT AAA GAA GAG TTA ATA AAA CTC GCA TAT AGC ATT 48 Met SerLeu Ile Ser Lys Glu Glu Leu Ile Lys Leu Ala Tyr Ser Ile 1 5 10 15 AGACCA AGA GAA AAT GAG TAT AAA ACT ATA CTA ACT AAT TTA GAC GAA 96 Arg ProArg Glu Asn Glu Tyr Lys Thr Ile Leu Thr Asn Leu Asp Glu 20 25 30 TAT AATAAG TTA ACT ACA AAC AAT AAT GAA AAT AAA TAT TTG CAA TTA 144 Tyr Asn LysLeu Thr Thr Asn Asn Asn Glu Asn Lys Tyr Leu Gln Leu 35 40 45 AAA AAA CTAAAT GAA TCA ATT GAT GTT TTT ATG AAT AAA TAT AAA ACT 192 Lys Lys Leu AsnGlu Ser Ile Asp Val Phe Met Asn Lys Tyr Lys Thr 50 55 60 TCA AGC AGA AATAGA GCA CTC TCT AAT CTA AAA AAA GAT ATA TTA AAA 240 Ser Ser Arg Asn ArgAla Leu Ser Asn Leu Lys Lys Asp Ile Leu Lys 65 70 75 80 GAA GTA ATT CTTATT AAA AAT TCC AAT ACA AGC CCT GTA GAA AAA AAT 288 Glu Val Ile Leu IleLys Asn Ser Asn Thr Ser Pro Val Glu Lys Asn 85 90 95 TTA CAT TTT GTA TGGATA GGT GGA GAA GTC AGT GAT ATT GCT CTT GAA 336 Leu His Phe Val Trp IleGly Gly Glu Val Ser Asp Ile Ala Leu Glu 100 105 110 TAC ATA AAA CAA TGGGCT GAT ATT AAT GCA GAA TAT AAT ATT AAA CTG 384 Tyr Ile Lys Gln Trp AlaAsp Ile Asn Ala Glu Tyr Asn Ile Lys Leu 115 120 125 TGG TAT GAT AGT GAAGCA TTC TTA GTA AAT ACA CTA AAA AAG GCT ATA 432 Trp Tyr Asp Ser Glu AlaPhe Leu Val Asn Thr Leu Lys Lys Ala Ile 130 135 140 GTT GAA TCT TCT ACCACT GAA GCA TTA CAG CTA CTA GAG GAA GAG ATT 480 Val Glu Ser Ser Thr ThrGlu Ala Leu Gln Leu Leu Glu Glu Glu Ile 145 150 155 160 CAA AAT CCT CAATTT GAT AAT ATG AAA TTT TAC AAA AAA AGG ATG GAA 528 Gln Asn Pro Gln PheAsp Asn Met Lys Phe Tyr Lys Lys Arg Met Glu 165 170 175 TTT ATA TAT GATAGA CAA AAA AGG TTT ATA AAT TAT TAT AAA TCT CAA 576 Phe Ile Tyr Asp ArgGln Lys Arg Phe Ile Asn Tyr Tyr Lys Ser Gln 180 185 190 ATC AAT AAA CCTACA GTA CCT ACA ATA GAT GAT ATT ATA AAG TCT CAT 624 Ile Asn Lys Pro ThrVal Pro Thr Ile Asp Asp Ile Ile Lys Ser His 195 200 205 CTA GTA TCT GAATAT AAT AGA GAT GAA ACT GTA TTA GAA TCA TAT AGA 672 Leu Val Ser Glu TyrAsn Arg Asp Glu Thr Val Leu Glu Ser Tyr Arg 210 215 220 ACA AAT TCT TTGAGA AAA ATA AAT AGT AAT CAT GGG ATA GAT ATC AGG 720 Thr Asn Ser Leu ArgLys Ile Asn Ser Asn His Gly Ile Asp Ile Arg 225 230 235 240 GCT AAT AGTTTG TTT ACA GAA CAA GAG TTA TTA AAT ATT TAT AGT CAG 768 Ala Asn Ser LeuPhe Thr Glu Gln Glu Leu Leu Asn Ile Tyr Ser Gln 245 250 255 GAG TTG TTAAAT CGT GGA AAT TTA GCT GCA GCA TCT GAC ATA GTA AGA 816 Glu Leu Leu AsnArg Gly Asn Leu Ala Ala Ala Ser Asp Ile Val Arg 260 265 270 TTA TTA GCCCTA AAA AAT TTT GGC GGA GTA TAT TTA GAT GTT GAT ATG 864 Leu Leu Ala LeuLys Asn Phe Gly Gly Val Tyr Leu Asp Val Asp Met 275 280 285 CTT CCA GGTATT CAC TCT GAT TTA TTT AAA ACA ATA TCT AGA CCT AGC 912 Leu Pro Gly IleHis Ser Asp Leu Phe Lys Thr Ile Ser Arg Pro Ser 290 295 300 TCT ATT GGACTA GAC CGT TGG GAA ATG ATA AAA TTA GAG GCT ATT ATG 960 Ser Ile Gly LeuAsp Arg Trp Glu Met Ile Lys Leu Glu Ala Ile Met 305 310 315 320 AAG TATAAA AAA TAT ATA AAT AAT TAT ACA TCA GAA AAC TTT GAT AAA 1008 Lys Tyr LysLys Tyr Ile Asn Asn Tyr Thr Ser Glu Asn Phe Asp Lys 325 330 335 CTT GATCAA CAA TTA AAA GAT AAT TTT AAA CTC ATT ATA GAA AGT AAA 1056 Leu Asp GlnGln Leu Lys Asp Asn Phe Lys Leu Ile Ile Glu Ser Lys 340 345 350 AGT GAAAAA TCT GAG ATA TTT TCT AAA TTA GAA AAT TTA AAT GTA TCT 1104 Ser Glu LysSer Glu Ile Phe Ser Lys Leu Glu Asn Leu Asn Val Ser 355 360 365 GAT CTTGAA ATT AAA ATA GCT TTC GCT TTA GGC AGT GTT ATA AAT CAA 1152 Asp Leu GluIle Lys Ile Ala Phe Ala Leu Gly Ser Val Ile Asn Gln 370 375 380 GCC TTGATA TCA AAA CAA GGT TCA TAT CTT ACT AAC CTA GTA ATA GAA 1200 Ala Leu IleSer Lys Gln Gly Ser Tyr Leu Thr Asn Leu Val Ile Glu 385 390 395 400 CAAGTA AAA AAT AGA TAT CAA TTT TTA AAC CAA CAC CTT AAC CCA GCC 1248 Gln ValLys Asn Arg Tyr Gln Phe Leu Asn Gln His Leu Asn Pro Ala 405 410 415 ATAGAG TCT GAT AAT AAC TTC ACA GAT ACT ACT AAA ATT TTT CAT GAT 1296 Ile GluSer Asp Asn Asn Phe Thr Asp Thr Thr Lys Ile Phe His Asp 420 425 430 TCATTA TTT AAT TCA GCT ACC GCA GAA AAC TCT ATG TTT TTA ACA AAA 1344 Ser LeuPhe Asn Ser Ala Thr Ala Glu Asn Ser Met Phe Leu Thr Lys 435 440 445 ATAGCA CCA TAC TTA CAA GTA GGT TTT ATG CCA GAA GCT CGC TCC ACA 1392 Ile AlaPro Tyr Leu Gln Val Gly Phe Met Pro Glu Ala Arg Ser Thr 450 455 460 ATAAGT TTA AGT GGT CCA GGA GCT TAT GCG TCA GCT TAC TAT GAT TTC 1440 Ile SerLeu Ser Gly Pro Gly Ala Tyr Ala Ser Ala Tyr Tyr Asp Phe 465 470 475 480ATA AAT TTA CAA GAA AAT ACT ATA GAA AAA ACT TTA AAA GCA TCA GAT 1488 IleAsn Leu Gln Glu Asn Thr Ile Glu Lys Thr Leu Lys Ala Ser Asp 485 490 495TTA ATA GAA TTT AAA TTC CCA GAA AAT AAT CTA TCT CAA TTG ACA GAA 1536 LeuIle Glu Phe Lys Phe Pro Glu Asn Asn Leu Ser Gln Leu Thr Glu 500 505 510CAA GAA ATA AAT AGT CTA TGG AGC TTT GAT CAA GCA AGT GCA AAA TAT 1584 GlnGlu Ile Asn Ser Leu Trp Ser Phe Asp Gln Ala Ser Ala Lys Tyr 515 520 525CAA TTT GAG AAA TAT GTA AGA GAT TAT ACT GGT GGA TCT CTT TCT GAA 1632 GlnPhe Glu Lys Tyr Val Arg Asp Tyr Thr Gly Gly Ser Leu Ser Glu 530 535 540GAC AAT GGG GTA GAC TTT AAT AAA AAT ACT GCC CTC GAC AAA AAC TAT 1680 AspAsn Gly Val Asp Phe Asn Lys Asn Thr Ala Leu Asp Lys Asn Tyr 545 550 555560 TTA TTA AAT AAT AAA ATT CCA TCA AAC AAT GTA GAA GAA GCT GGA AGT 1728Leu Leu Asn Asn Lys Ile Pro Ser Asn Asn Val Glu Glu Ala Gly Ser 565 570575 AAA AAT TAT GTT CAT TAT ATC ATA CAG TTA CAA GGA GAT GAT ATA AGT 1776Lys Asn Tyr Val His Tyr Ile Ile Gln Leu Gln Gly Asp Asp Ile Ser 580 585590 TAT GAA GCA ACA TGC AAT TTA TTT TCT AAA AAT CCT AAA AAT AGT ATT 1824Tyr Glu Ala Thr Cys Asn Leu Phe Ser Lys Asn Pro Lys Asn Ser Ile 595 600605 ATT ATA CAA CGA AAT ATG AAT GAA AGT GCA AAA AGC TAC TTT TTA AGT 1872Ile Ile Gln Arg Asn Met Asn Glu Ser Ala Lys Ser Tyr Phe Leu Ser 610 615620 GAT GAT GGA GAA TCT ATT TTA GAA TTA AAT AAA TAT AGG ATA CCT GAA 1920Asp Asp Gly Glu Ser Ile Leu Glu Leu Asn Lys Tyr Arg Ile Pro Glu 625 630635 640 AGA TTA AAA AAT AAG GAA AAA GTA AAA GTA ACC TTT ATT GGA CAT GGT1968 Arg Leu Lys Asn Lys Glu Lys Val Lys Val Thr Phe Ile Gly His Gly 645650 655 AAA GAT GAA TTC AAC ACA AGC GAA TTT GCT AGA TTA AGT GTA GAT TCA2016 Lys Asp Glu Phe Asn Thr Ser Glu Phe Ala Arg Leu Ser Val Asp Ser 660665 670 CTT TCC AAT GAG ATA AGT TCA TTT TTA GAT ACC ATA AAA TTA GAT ATA2064 Leu Ser Asn Glu Ile Ser Ser Phe Leu Asp Thr Ile Lys Leu Asp Ile 675680 685 TCA CCT AAA AAT GTA GAA GTA AAC TTA CTT GGA TGT AAT ATG TTT AGT2112 Ser Pro Lys Asn Val Glu Val Asn Leu Leu Gly Cys Asn Met Phe Ser 690695 700 TAT GAT TTT AAT GTT GAA GAA ACT TAT CCT GGG AAG TTG CTA TTA AGT2160 Tyr Asp Phe Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Ser 705710 715 720 ATT ATG GAC AAA ATT ACT TCC ACT TTA CCT GAT GTA AAT AAA AATTCT 2208 Ile Met Asp Lys Ile Thr Ser Thr Leu Pro Asp Val Asn Lys Asn Ser725 730 735 ATT ACT ATA GGA GCA AAT CAA TAT GAA GTA AGA ATT AAT AGT GAGGGA 2256 Ile Thr Ile Gly Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly740 745 750 AGA AAA GAA CTT CTG GCT CAC TCA GGT AAA TGG ATA AAT AAA GAAGAA 2304 Arg Lys Glu Leu Leu Ala His Ser Gly Lys Trp Ile Asn Lys Glu Glu755 760 765 GCT ATT ATG AGC GAT TTA TCT AGT AAA GAA TAC ATT TTT TTT GATTCT 2352 Ala Ile Met Ser Asp Leu Ser Ser Lys Glu Tyr Ile Phe Phe Asp Ser770 775 780 ATA GAT AAT AAG CTA AAA GCA AAG TCC AAG AAT ATT CCA GGA TTAGCA 2400 Ile Asp Asn Lys Leu Lys Ala Lys Ser Lys Asn Ile Pro Gly Leu Ala785 790 795 800 TCA ATA TCA GAA GAT ATA AAA ACA TTA TTA CTT GAT GCA AGTGTT AGT 2448 Ser Ile Ser Glu Asp Ile Lys Thr Leu Leu Leu Asp Ala Ser ValSer 805 810 815 CCT GAT ACA AAA TTT ATT TTA AAT AAT CTT AAG CTT AAT ATTGAA TCT 2496 Pro Asp Thr Lys Phe Ile Leu Asn Asn Leu Lys Leu Asn Ile GluSer 820 825 830 TCT ATT GGG GAT TAC ATT TAT TAT GAA AAA TTA GAG CCT GTTAAA AAT 2544 Ser Ile Gly Asp Tyr Ile Tyr Tyr Glu Lys Leu Glu Pro Val LysAsn 835 840 845 ATA ATT CAC AAT TCT ATA GAT GAT TTA ATA GAT GAG TTC AATCTA CTT 2592 Ile Ile His Asn Ser Ile Asp Asp Leu Ile Asp Glu Phe Asn LeuLeu 850 855 860 GAA AAT GTA TCT GAT GAA TTA TAT GAA TTA AAA AAA TTA AATAAT CTA 2640 Glu Asn Val Ser Asp Glu Leu Tyr Glu Leu Lys Lys Leu Asn AsnLeu 865 870 875 880 GAT GAG AAG TAT TTA ATA TCT TTT GAA GAT ATC TCA AAAAAT AAT TCA 2688 Asp Glu Lys Tyr Leu Ile Ser Phe Glu Asp Ile Ser Lys AsnAsn Ser 885 890 895 ACT TAC TCT GTA AGA TTT ATT AAC AAA AGT AAT GGT GAGTCA GTT TAT 2736 Thr Tyr Ser Val Arg Phe Ile Asn Lys Ser Asn Gly Glu SerVal Tyr 900 905 910 GTA GAA ACA GAA AAA GAA ATT TTT TCA AAA TAT AGC GAACAT ATT ACA 2784 Val Glu Thr Glu Lys Glu Ile Phe Ser Lys Tyr Ser Glu HisIle Thr 915 920 925 AAA GAA ATA AGT ACT ATA AAG AAT AGT ATA ATT ACA GATGTT AAT GGT 2832 Lys Glu Ile Ser Thr Ile Lys Asn Ser Ile Ile Thr Asp ValAsn Gly 930 935 940 AAT TTA TTG GAT AAT ATA CAG TTA GAT CAT ACT TCT CAAGTT AAT ACA 2880 Asn Leu Leu Asp Asn Ile Gln Leu Asp His Thr Ser Gln ValAsn Thr 945 950 955 960 TTA AAC GCA GCA TTC TTT ATT CAA TCA TTA ATA GATTAT AGT AGC AAT 2928 Leu Asn Ala Ala Phe Phe Ile Gln Ser Leu Ile Asp TyrSer Ser Asn 965 970 975 AAA GAT GTA CTG AAT GAT TTA AGT ACC TCA GTT AAGGTT CAA CTT TAT 2976 Lys Asp Val Leu Asn Asp Leu Ser Thr Ser Val Lys ValGln Leu Tyr 980 985 990 GCT CAA CTA TTT AGT ACA GGT TTA AAT ACT ATA TATGAC TCT ATC CAA 3024 Ala Gln Leu Phe Ser Thr Gly Leu Asn Thr Ile Tyr AspSer Ile Gln 995 1000 1005 TTA GTA AAT TTA ATA TCA AAT GCA GTA AAT GATACT ATA AAT GTA CTA 3072 Leu Val Asn Leu Ile Ser Asn Ala Val Asn Asp ThrIle Asn Val Leu 1010 1015 1020 CCT ACA ATA ACA GAG GGG ATA CCT ATT GTATCT ACT ATA TTA GAC GGA 3120 Pro Thr Ile Thr Glu Gly Ile Pro Ile Val SerThr Ile Leu Asp Gly 1025 1030 1035 1040 ATA AAC TTA GGT GCA GCA ATT AAGGAA TTA CTA GAC GAA CAT GAC CCA 3168 Ile Asn Leu Gly Ala Ala Ile Lys GluLeu Leu Asp Glu His Asp Pro 1045 1050 1055 TTA CTA AAA AAA GAA TTA GAAGCT AAG GTG GGT GTT TTA GCA ATA AAT 3216 Leu Leu Lys Lys Glu Leu Glu AlaLys Val Gly Val Leu Ala Ile Asn 1060 1065 1070 ATG TCA TTA TCT ATA GCTGCA ACT GTA GCT TCA ATT GTT GGA ATA GGT 3264 Met Ser Leu Ser Ile Ala AlaThr Val Ala Ser Ile Val Gly Ile Gly 1075 1080 1085 GCT GAA GTT ACT ATTTTC TTA TTA CCT ATA GCT GGT ATA TCT GCA GGA 3312 Ala Glu Val Thr Ile PheLeu Leu Pro Ile Ala Gly Ile Ser Ala Gly 1090 1095 1100 ATA CCT TCA TTAGTT AAT AAT GAA TTA ATA TTG CAT GAT AAG GCA ACT 3360 Ile Pro Ser Leu ValAsn Asn Glu Leu Ile Leu His Asp Lys Ala Thr 1105 1110 1115 1120 TCA GTGGTA AAC TAT TTT AAT CAT TTG TCT GAA TCT AAA AAA TAT GGC 3408 Ser Val ValAsn Tyr Phe Asn His Leu Ser Glu Ser Lys Lys Tyr Gly 1125 1130 1135 CCTCTT AAA ACA GAA GAT GAT AAA ATT TTA GTT CCT ATT GAT GAT TTA 3456 Pro LeuLys Thr Glu Asp Asp Lys Ile Leu Val Pro Ile Asp Asp Leu 1140 1145 1150GTA ATA TCA GAA ATA GAT TTT AAT AAT AAT TCG ATA AAA CTA GGA ACA 3504 ValIle Ser Glu Ile Asp Phe Asn Asn Asn Ser Ile Lys Leu Gly Thr 1155 11601165 TGT AAT ATA TTA GCA ATG GAG GGG GGA TCA GGA CAC ACA GTG ACT GGT3552 Cys Asn Ile Leu Ala Met Glu Gly Gly Ser Gly His Thr Val Thr Gly1170 1175 1180 AAT ATA GAT CAC TTT TTC TCA TCT CCA TCT ATA AGT TCT CATATT CCT 3600 Asn Ile Asp His Phe Phe Ser Ser Pro Ser Ile Ser Ser His IlePro 1185 1190 1195 1200 TCA TTA TCA ATT TAT TCT GCA ATA GGT ATA GAA ACAGAA AAT CTA GAT 3648 Ser Leu Ser Ile Tyr Ser Ala Ile Gly Ile Glu Thr GluAsn Leu Asp 1205 1210 1215 TTT TCA AAA AAA ATA ATG ATG TTA CCT AAT GCTCCT TCA AGA GTG TTT 3696 Phe Ser Lys Lys Ile Met Met Leu Pro Asn Ala ProSer Arg Val Phe 1220 1225 1230 TGG TGG GAA ACT GGA GCA GTT CCA GGT TTAAGA TCA TTG GAA AAT GAC 3744 Trp Trp Glu Thr Gly Ala Val Pro Gly Leu ArgSer Leu Glu Asn Asp 1235 1240 1245 GGA ACT AGA TTA CTT GAT TCA ATA AGAGAT TTA TAC CCA GGT AAA TTT 3792 Gly Thr Arg Leu Leu Asp Ser Ile Arg AspLeu Tyr Pro Gly Lys Phe 1250 1255 1260 TAC TGG AGA TTC TAT GCT TTT TTCGAT TAT GCA ATA ACT ACA TTA AAA 3840 Tyr Trp Arg Phe Tyr Ala Phe Phe AspTyr Ala Ile Thr Thr Leu Lys 1265 1270 1275 1280 CCA GTT TAT GAA GAC ACTAAT ATT AAA ATT AAA CTA GAT AAA GAT ACT 3888 Pro Val Tyr Glu Asp Thr AsnIle Lys Ile Lys Leu Asp Lys Asp Thr 1285 1290 1295 AGA AAC TTC ATA ATGCCA ACT ATA ACT ACT AAC GAA ATT AGA AAC AAA 3936 Arg Asn Phe Ile Met ProThr Ile Thr Thr Asn Glu Ile Arg Asn Lys 1300 1305 1310 TTA TCT TAT TCATTT GAT GGA GCA GGA GGA ACT TAC TCT TTA TTA TTA 3984 Leu Ser Tyr Ser PheAsp Gly Ala Gly Gly Thr Tyr Ser Leu Leu Leu 1315 1320 1325 TCT TCA TATCCA ATA TCA ACG AAT ATA AAT TTA TCT AAA GAT GAT TTA 4032 Ser Ser Tyr ProIle Ser Thr Asn Ile Asn Leu Ser Lys Asp Asp Leu 1330 1335 1340 TGG ATATTT AAT ATT GAT AAT GAA GTA AGA GAA ATA TCT ATA GAA AAT 4080 Trp Ile PheAsn Ile Asp Asn Glu Val Arg Glu Ile Ser Ile Glu Asn 1345 1350 1355 1360GGT ACT ATT AAA AAA GGA AAG TTA ATA AAA GAT GTT TTA AGT AAA ATT 4128 GlyThr Ile Lys Lys Gly Lys Leu Ile Lys Asp Val Leu Ser Lys Ile 1365 13701375 GAT ATA AAT AAA AAT AAA CTT ATT ATA GGC AAT CAA ACA ATA GAT TTT4176 Asp Ile Asn Lys Asn Lys Leu Ile Ile Gly Asn Gln Thr Ile Asp Phe1380 1385 1390 TCA GGC GAT ATA GAT AAT AAA GAT AGA TAT ATA TTC TTG ACTTGT GAG 4224 Ser Gly Asp Ile Asp Asn Lys Asp Arg Tyr Ile Phe Leu Thr CysGlu 1395 1400 1405 TTA GAT GAT AAA ATT AGT TTA ATA ATA GAA ATA AAT CTTGTT GCA AAA 4272 Leu Asp Asp Lys Ile Ser Leu Ile Ile Glu Ile Asn Leu ValAla Lys 1410 1415 1420 TCT TAT AGT TTG TTA TTG TCT GGG GAT AAA AAT TATTTG ATA TCC AAT 4320 Ser Tyr Ser Leu Leu Leu Ser Gly Asp Lys Asn Tyr LeuIle Ser Asn 1425 1430 1435 1440 TTA TCT AAT ACT ATT GAG AAA ATC AAT ACTTTA GGC CTA GAT AGT AAA 4368 Leu Ser Asn Thr Ile Glu Lys Ile Asn Thr LeuGly Leu Asp Ser Lys 1445 1450 1455 AAT ATA GCG TAC AAT TAC ACT GAT GAATCT AAT AAT AAA TAT TTT GGA 4416 Asn Ile Ala Tyr Asn Tyr Thr Asp Glu SerAsn Asn Lys Tyr Phe Gly 1460 1465 1470 GCT ATA TCT AAA ACA AGT CAA AAAAGC ATA ATA CAT TAT AAA AAA GAC 4464 Ala Ile Ser Lys Thr Ser Gln Lys SerIle Ile His Tyr Lys Lys Asp 1475 1480 1485 AGT AAA AAT ATA TTA GAA TTTTAT AAT GAC AGT ACA TTA GAA TTT AAC 4512 Ser Lys Asn Ile Leu Glu Phe TyrAsn Asp Ser Thr Leu Glu Phe Asn 1490 1495 1500 AGT AAA GAT TTT ATT GCTGAA GAT ATA AAT GTA TTT ATG AAA GAT GAT 4560 Ser Lys Asp Phe Ile Ala GluAsp Ile Asn Val Phe Met Lys Asp Asp 1505 1510 1515 1520 ATT AAT ACT ATAACA GGA AAA TAC TAT GTT GAT AAT AAT ACT GAT AAA 4608 Ile Asn Thr Ile ThrGly Lys Tyr Tyr Val Asp Asn Asn Thr Asp Lys 1525 1530 1535 AGT ATA GATTTC TCT ATT TCT TTA GTT AGT AAA AAT CAA GTA AAA GTA 4656 Ser Ile Asp PheSer Ile Ser Leu Val Ser Lys Asn Gln Val Lys Val 1540 1545 1550 AAT GGATTA TAT TTA AAT GAA TCC GTA TAC TCA TCT TAC CTT GAT TTT 4704 Asn Gly LeuTyr Leu Asn Glu Ser Val Tyr Ser Ser Tyr Leu Asp Phe 1555 1560 1565 GTGAAA AAT TCA GAT GGA CAC CAT AAT ACT TCT AAT TTT ATG AAT TTA 4752 Val LysAsn Ser Asp Gly His His Asn Thr Ser Asn Phe Met Asn Leu 1570 1575 1580TTT TTG GAC AAT ATA AGT TTC TGG AAA TTG TTT GGG TTT GAA AAT ATA 4800 PheLeu Asp Asn Ile Ser Phe Trp Lys Leu Phe Gly Phe Glu Asn Ile 1585 15901595 1600 AAT TTT GTA ATC GAT AAA TAC TTT ACC CTT GTT GGT AAA ACT AATCTT 4848 Asn Phe Val Ile Asp Lys Tyr Phe Thr Leu Val Gly Lys Thr Asn Leu1605 1610 1615 GGA TAT GTA GAA TTT ATT TGT GAC AAT AAT AAA AAT ATA GATATA TAT 4896 Gly Tyr Val Glu Phe Ile Cys Asp Asn Asn Lys Asn Ile Asp IleTyr 1620 1625 1630 TTT GGT GAA TGG AAA ACA TCG TCA TCT AAA AGC ACT ATATTT AGC GGA 4944 Phe Gly Glu Trp Lys Thr Ser Ser Ser Lys Ser Thr Ile PheSer Gly 1635 1640 1645 AAT GGT AGA AAT GTT GTA GTA GAG CCT ATA TAT AATCCT GAT ACG GGT 4992 Asn Gly Arg Asn Val Val Val Glu Pro Ile Tyr Asn ProAsp Thr Gly 1650 1655 1660 GAA GAT ATA TCT ACT TCA CTA GAT TTT TCC TATGAA CCT CTC TAT GGA 5040 Glu Asp Ile Ser Thr Ser Leu Asp Phe Ser Tyr GluPro Leu Tyr Gly 1665 1670 1675 1680 ATA GAT AGA TAT ATA AAT AAA GTA TTGATA GCA CCT GAT TTA TAT ACA 5088 Ile Asp Arg Tyr Ile Asn Lys Val Leu IleAla Pro Asp Leu Tyr Thr 1685 1690 1695 AGT TTA ATA AAT ATT AAT ACC AATTAT TAT TCA AAT GAG TAC TAC CCT 5136 Ser Leu Ile Asn Ile Asn Thr Asn TyrTyr Ser Asn Glu Tyr Tyr Pro 1700 1705 1710 GAG ATT ATA GTT CTT AAC CCAAAT ACA TTC CAC AAA AAA GTA AAT ATA 5184 Glu Ile Ile Val Leu Asn Pro AsnThr Phe His Lys Lys Val Asn Ile 1715 1720 1725 AAT TTA GAT AGT TCT TCTTTT GAG TAT AAA TGG TCT ACA GAA GGA AGT 5232 Asn Leu Asp Ser Ser Ser PheGlu Tyr Lys Trp Ser Thr Glu Gly Ser 1730 1735 1740 GAC TTT ATT TTA GTTAGA TAC TTA GAA GAA AGT AAT AAA AAA ATA TTA 5280 Asp Phe Ile Leu Val ArgTyr Leu Glu Glu Ser Asn Lys Lys Ile Leu 1745 1750 1755 1760 CAA AAA ATAAGA ATC AAA GGT ATC TTA TCT AAT ACT CAA TCA TTT AAT 5328 Gln Lys Ile ArgIle Lys Gly Ile Leu Ser Asn Thr Gln Ser Phe Asn 1765 1770 1775 AAA ATGAGT ATA GAT TTT AAA GAT ATT AAA AAA CTA TCA TTA GGA TAT 5376 Lys Met SerIle Asp Phe Lys Asp Ile Lys Lys Leu Ser Leu Gly Tyr 1780 1785 1790 ATAATG AGT AAT TTT AAA TCA TTT AAT TCT GAA AAT GAA TTA GAT AGA 5424 Ile MetSer Asn Phe Lys Ser Phe Asn Ser Glu Asn Glu Leu Asp Arg 1795 1800 1805GAT CAT TTA GGA TTT AAA ATA ATA GAT AAT AAA ACT TAT TAC TAT GAT 5472 AspHis Leu Gly Phe Lys Ile Ile Asp Asn Lys Thr Tyr Tyr Tyr Asp 1810 18151820 GAA GAT AGT AAA TTA GTT AAA GGA TTA ATC AAT ATA AAT AAT TCA TTA5520 Glu Asp Ser Lys Leu Val Lys Gly Leu Ile Asn Ile Asn Asn Ser Leu1825 1830 1835 1840 TTC TAT TTT GAT CCT ATA GAA TTT AAC TTA GTA ACT GGATGG CAA ACT 5568 Phe Tyr Phe Asp Pro Ile Glu Phe Asn Leu Val Thr Gly TrpGln Thr 1845 1850 1855 ATC AAT GGT AAA AAA TAT TAT TTT GAT ATA AAT ACTGGA GCA GCT TTA 5616 Ile Asn Gly Lys Lys Tyr Tyr Phe Asp Ile Asn Thr GlyAla Ala Leu 1860 1865 1870 ACT AGT TAT AAA ATT ATT AAT GGT AAA CAC TTTTAT TTT AAT AAT GAT 5664 Thr Ser Tyr Lys Ile Ile Asn Gly Lys His Phe TyrPhe Asn Asn Asp 1875 1880 1885 GGT GTG ATG CAG TTG GGA GTA TTT AAA GGACCT GAT GGA TTT GAA TAT 5712 Gly Val Met Gln Leu Gly Val Phe Lys Gly ProAsp Gly Phe Glu Tyr 1890 1895 1900 TTT GCA CCT GCC AAT ACT CAA AAT AATAAC ATA GAA GGT CAG GCT ATA 5760 Phe Ala Pro Ala Asn Thr Gln Asn Asn AsnIle Glu Gly Gln Ala Ile 1905 1910 1915 1920 GTT TAT CAA AGT AAA TTC TTAACT TTG AAT GGC AAA AAA TAT TAT TTT 5808 Val Tyr Gln Ser Lys Phe Leu ThrLeu Asn Gly Lys Lys Tyr Tyr Phe 1925 1930 1935 GAT AAT AAC TCA AAA GCAGTC ACT GGA TGG AGA ATT ATT AAC AAT GAG 5856 Asp Asn Asn Ser Lys Ala ValThr Gly Trp Arg Ile Ile Asn Asn Glu 1940 1945 1950 AAA TAT TAC TTT AATCCT AAT AAT GCT ATT GCT GCA GTC GGA TTG CAA 5904 Lys Tyr Tyr Phe Asn ProAsn Asn Ala Ile Ala Ala Val Gly Leu Gln 1955 1960 1965 GTA ATT GAC AATAAT AAG TAT TAT TTC AAT CCT GAC ACT GCT ATC ATC 5952 Val Ile Asp Asn AsnLys Tyr Tyr Phe Asn Pro Asp Thr Ala Ile Ile 1970 1975 1980 TCA AAA GGTTGG CAG ACT GTT AAT GGT AGT AGA TAC TAC TTT GAT ACT 6000 Ser Lys Gly TrpGln Thr Val Asn Gly Ser Arg Tyr Tyr Phe Asp Thr 1985 1990 1995 2000 GATACC GCT ATT GCC TTT AAT GGT TAT AAA ACT ATT GAT GGT AAA CAC 6048 Asp ThrAla Ile Ala Phe Asn Gly Tyr Lys Thr Ile Asp Gly Lys His 2005 2010 2015TTT TAT TTT GAT AGT GAT TGT GTA GTG AAA ATA GGT GTG TTT AGT ACC 6096 PheTyr Phe Asp Ser Asp Cys Val Val Lys Ile Gly Val Phe Ser Thr 2020 20252030 TCT AAT GGA TTT GAA TAT TTT GCA CCT GCT AAT ACT TAT AAT AAT AAC6144 Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Tyr Asn Asn Asn2035 2040 2045 ATA GAA GGT CAG GCT ATA GTT TAT CAA AGT AAA TTC TTA ACTTTG AAT 6192 Ile Glu Gly Gln Ala Ile Val Tyr Gln Ser Lys Phe Leu Thr LeuAsn 2050 2055 2060 GGT AAA AAA TAT TAC TTT GAT AAT AAC TCA AAA GCA GTTACC GGA TTG 6240 Gly Lys Lys Tyr Tyr Phe Asp Asn Asn Ser Lys Ala Val ThrGly Leu 2065 2070 2075 2080 CAA ACT ATT GAT AGT AAA AAA TAT TAC TTT AATACT AAC ACT GCT GAA 6288 Gln Thr Ile Asp Ser Lys Lys Tyr Tyr Phe Asn ThrAsn Thr Ala Glu 2085 2090 2095 GCA GCT ACT GGA TGG CAA ACT ATT GAT GGTAAA AAA TAT TAC TTT AAT 6336 Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly LysLys Tyr Tyr Phe Asn 2100 2105 2110 ACT AAC ACT GCT GAA GCA GCT ACT GGATGG CAA ACT ATT GAT GGT AAA 6384 Thr Asn Thr Ala Glu Ala Ala Thr Gly TrpGln Thr Ile Asp Gly Lys 2115 2120 2125 AAA TAT TAC TTT AAT ACT AAC ACTGCT ATA GCT TCA ACT GGT TAT ACA 6432 Lys Tyr Tyr Phe Asn Thr Asn Thr AlaIle Ala Ser Thr Gly Tyr Thr 2130 2135 2140 ATT ATT AAT GGT AAA CAT TTTTAT TTT AAT ACT GAT GGT ATT ATG CAG 6480 Ile Ile Asn Gly Lys His Phe TyrPhe Asn Thr Asp Gly Ile Met Gln 2145 2150 2155 2160 ATA GGA GTG TTT AAAGGA CCT AAT GGA TTT GAA TAT TTT GCA CCT GCT 6528 Ile Gly Val Phe Lys GlyPro Asn Gly Phe Glu Tyr Phe Ala Pro Ala 2165 2170 2175 AAT ACG GAT GCTAAC AAC ATA GAA GGT CAA GCT ATA CTT TAC CAA AAT 6576 Asn Thr Asp Ala AsnAsn Ile Glu Gly Gln Ala Ile Leu Tyr Gln Asn 2180 2185 2190 GAA TTC TTAACT TTG AAT GGT AAA AAA TAT TAC TTT GGT AGT GAC TCA 6624 Glu Phe Leu ThrLeu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser 2195 2200 2205 AAA GCAGTT ACT GGA TGG AGA ATT ATT AAC AAT AAG AAA TAT TAC TTT 6672 Lys Ala ValThr Gly Trp Arg Ile Ile Asn Asn Lys Lys Tyr Tyr Phe 2210 2215 2220 AATCCT AAT AAT GCT ATT GCT GCA ATT CAT CTA TGC ACT ATA AAT AAT 6720 Asn ProAsn Asn Ala Ile Ala Ala Ile His Leu Cys Thr Ile Asn Asn 2225 2230 22352240 GAC AAG TAT TAC TTT AGT TAT GAT GGA ATT CTT CAA AAT GGA TAT ATT6768 Asp Lys Tyr Tyr Phe Ser Tyr Asp Gly Ile Leu Gln Asn Gly Tyr Ile2245 2250 2255 ACT ATT GAA AGA AAT AAT TTC TAT TTT GAT GCT AAT AAT GAATCT AAA 6816 Thr Ile Glu Arg Asn Asn Phe Tyr Phe Asp Ala Asn Asn Glu SerLys 2260 2265 2270 ATG GTA ACA GGA GTA TTT AAA GGA CCT AAT GGA TTT GAGTAT TTT GCA 6864 Met Val Thr Gly Val Phe Lys Gly Pro Asn Gly Phe Glu TyrPhe Ala 2275 2280 2285 CCT GCT AAT ACT CAC AAT AAT AAC ATA GAA GGT CAGGCT ATA GTT TAC 6912 Pro Ala Asn Thr His Asn Asn Asn Ile Glu Gly Gln AlaIle Val Tyr 2290 2295 2300 CAG AAC AAA TTC TTA ACT TTG AAT GGC AAA AAATAT TAT TTT GAT AAT 6960 Gln Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys TyrTyr Phe Asp Asn 2305 2310 2315 2320 GAC TCA AAA GCA GTT ACT GGA TGG CAAACC ATT GAT GGT AAA AAA TAT 7008 Asp Ser Lys Ala Val Thr Gly Trp Gln ThrIle Asp Gly Lys Lys Tyr 2325 2330 2335 TAC TTT AAT CTT AAC ACT GCT GAAGCA GCT ACT GGA TGG CAA ACT ATT 7056 Tyr Phe Asn Leu Asn Thr Ala Glu AlaAla Thr Gly Trp Gln Thr Ile 2340 2345 2350 GAT GGT AAA AAA TAT TAC TTTAAT CTT AAC ACT GCT GAA GCA GCT ACT 7104 Asp Gly Lys Lys Tyr Tyr Phe AsnLeu Asn Thr Ala Glu Ala Ala Thr 2355 2360 2365 GGA TGG CAA ACT ATT GATGGT AAA AAA TAT TAC TTT AAT ACT AAC ACT 7152 Gly Trp Gln Thr Ile Asp GlyLys Lys Tyr Tyr Phe Asn Thr Asn Thr 2370 2375 2380 TTC ATA GCC TCA ACTGGT TAT ACA AGT ATT AAT GGT AAA CAT TTT TAT 7200 Phe Ile Ala Ser Thr GlyTyr Thr Ser Ile Asn Gly Lys His Phe Tyr 2385 2390 2395 2400 TTT AAT ACTGAT GGT ATT ATG CAG ATA GGA GTG TTT AAA GGA CCT AAT 7248 Phe Asn Thr AspGly Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asn 2405 2410 2415 GGA TTTGAA TAC TTT GCA CCT GCT AAT ACG GAT GCT AAC AAC ATA GAA 7296 Gly Phe GluTyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu 2420 2425 2430 GGTCAA GCT ATA CTT TAC CAA AAT AAA TTC TTA ACT TTG AAT GGT AAA 7344 Gly GlnAla Ile Leu Tyr Gln Asn Lys Phe Leu Thr Leu Asn Gly Lys 2435 2440 2445AAA TAT TAC TTT GGT AGT GAC TCA AAA GCA GTT ACC GGA CTG CGA ACT 7392 LysTyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly Leu Arg Thr 2450 24552460 ATT GAT GGT AAA AAA TAT TAC TTT AAT ACT AAC ACT GCT GTT GCA GTT7440 Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Val Ala Val2465 2470 2475 2480 ACT GGA TGG CAA ACT ATT AAT GGT AAA AAA TAC TAC TTTAAT ACT AAC 7488 Thr Gly Trp Gln Thr Ile Asn Gly Lys Lys Tyr Tyr Phe AsnThr Asn 2485 2490 2495 ACT TCT ATA GCT TCA ACT GGT TAT ACA ATT ATT AGTGGT AAA CAT TTT 7536 Thr Ser Ile Ala Ser Thr Gly Tyr Thr Ile Ile Ser GlyLys His Phe 2500 2505 2510 TAT TTT AAT ACT GAT GGT ATT ATG CAG ATA GGAGTG TTT AAA GGA CCT 7584 Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly ValPhe Lys Gly Pro 2515 2520 2525 GAT GGA TTT GAA TAC TTT GCA CCT GCT AATACA GAT GCT AAC AAT ATA 7632 Asp Gly Phe Glu Tyr Phe Ala Pro Ala Asn ThrAsp Ala Asn Asn Ile 2530 2535 2540 GAA GGT CAA GCT ATA CGT TAT CAA AATAGA TTC CTA TAT TTA CAT GAC 7680 Glu Gly Gln Ala Ile Arg Tyr Gln Asn ArgPhe Leu Tyr Leu His Asp 2545 2550 2555 2560 AAT ATA TAT TAT TTT GGT AATAAT TCA AAA GCG GCT ACT GGT TGG GTA 7728 Asn Ile Tyr Tyr Phe Gly Asn AsnSer Lys Ala Ala Thr Gly Trp Val 2565 2570 2575 ACT ATT GAT GGT AAT AGATAT TAC TTC GAG CCT AAT ACA GCT ATG GGT 7776 Thr Ile Asp Gly Asn Arg TyrTyr Phe Glu Pro Asn Thr Ala Met Gly 2580 2585 2590 GCG AAT GGT TAT AAAACT ATT GAT AAT AAA AAT TTT TAC TTT AGA AAT 7824 Ala Asn Gly Tyr Lys ThrIle Asp Asn Lys Asn Phe Tyr Phe Arg Asn 2595 2600 2605 GGT TTA CCT CAGATA GGA GTG TTT AAA GGG TCT AAT GGA TTT GAA TAC 7872 Gly Leu Pro Gln IleGly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr 2610 2615 2620 TTT GCA CCTGCT AAT ACG GAT GCT AAC AAT ATA GAA GGT CAA GCT ATA 7920 Phe Ala Pro AlaAsn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile 2625 2630 2635 2640 CGTTAT CAA AAT AGA TTC CTA CAT TTA CTT GGA AAA ATA TAT TAC TTT 7968 Arg TyrGln Asn Arg Phe Leu His Leu Leu Gly Lys Ile Tyr Tyr Phe 2645 2650 2655GGT AAT AAT TCA AAA GCA GTT ACT GGA TGG CAA ACT ATT AAT GGT AAA 8016 GlyAsn Asn Ser Lys Ala Val Thr Gly Trp Gln Thr Ile Asn Gly Lys 2660 26652670 GTA TAT TAC TTT ATG CCT GAT ACT GCT ATG GCT GCA GCT GGT GGA CTT8064 Val Tyr Tyr Phe Met Pro Asp Thr Ala Met Ala Ala Ala Gly Gly Leu2675 2680 2685 TTC GAG ATT GAT GGT GTT ATA TAT TTC TTT GGT GTT GAT GGAGTA AAA 8112 Phe Glu Ile Asp Gly Val Ile Tyr Phe Phe Gly Val Asp Gly ValLys 2690 2695 2700 GCC CCT GGG ATA TAT GGC TAA 8133 Ala Pro Gly Ile TyrGly 2705 2710 2710 amino acids amino acid linear protein 6 Met Ser LeuIle Ser Lys Glu Glu Leu Ile Lys Leu Ala Tyr Ser Ile 1 5 10 15 Arg ProArg Glu Asn Glu Tyr Lys Thr Ile Leu Thr Asn Leu Asp Glu 20 25 30 Tyr AsnLys Leu Thr Thr Asn Asn Asn Glu Asn Lys Tyr Leu Gln Leu 35 40 45 Lys LysLeu Asn Glu Ser Ile Asp Val Phe Met Asn Lys Tyr Lys Thr 50 55 60 Ser SerArg Asn Arg Ala Leu Ser Asn Leu Lys Lys Asp Ile Leu Lys 65 70 75 80 GluVal Ile Leu Ile Lys Asn Ser Asn Thr Ser Pro Val Glu Lys Asn 85 90 95 LeuHis Phe Val Trp Ile Gly Gly Glu Val Ser Asp Ile Ala Leu Glu 100 105 110Tyr Ile Lys Gln Trp Ala Asp Ile Asn Ala Glu Tyr Asn Ile Lys Leu 115 120125 Trp Tyr Asp Ser Glu Ala Phe Leu Val Asn Thr Leu Lys Lys Ala Ile 130135 140 Val Glu Ser Ser Thr Thr Glu Ala Leu Gln Leu Leu Glu Glu Glu Ile145 150 155 160 Gln Asn Pro Gln Phe Asp Asn Met Lys Phe Tyr Lys Lys ArgMet Glu 165 170 175 Phe Ile Tyr Asp Arg Gln Lys Arg Phe Ile Asn Tyr TyrLys Ser Gln 180 185 190 Ile Asn Lys Pro Thr Val Pro Thr Ile Asp Asp IleIle Lys Ser His 195 200 205 Leu Val Ser Glu Tyr Asn Arg Asp Glu Thr ValLeu Glu Ser Tyr Arg 210 215 220 Thr Asn Ser Leu Arg Lys Ile Asn Ser AsnHis Gly Ile Asp Ile Arg 225 230 235 240 Ala Asn Ser Leu Phe Thr Glu GlnGlu Leu Leu Asn Ile Tyr Ser Gln 245 250 255 Glu Leu Leu Asn Arg Gly AsnLeu Ala Ala Ala Ser Asp Ile Val Arg 260 265 270 Leu Leu Ala Leu Lys AsnPhe Gly Gly Val Tyr Leu Asp Val Asp Met 275 280 285 Leu Pro Gly Ile HisSer Asp Leu Phe Lys Thr Ile Ser Arg Pro Ser 290 295 300 Ser Ile Gly LeuAsp Arg Trp Glu Met Ile Lys Leu Glu Ala Ile Met 305 310 315 320 Lys TyrLys Lys Tyr Ile Asn Asn Tyr Thr Ser Glu Asn Phe Asp Lys 325 330 335 LeuAsp Gln Gln Leu Lys Asp Asn Phe Lys Leu Ile Ile Glu Ser Lys 340 345 350Ser Glu Lys Ser Glu Ile Phe Ser Lys Leu Glu Asn Leu Asn Val Ser 355 360365 Asp Leu Glu Ile Lys Ile Ala Phe Ala Leu Gly Ser Val Ile Asn Gln 370375 380 Ala Leu Ile Ser Lys Gln Gly Ser Tyr Leu Thr Asn Leu Val Ile Glu385 390 395 400 Gln Val Lys Asn Arg Tyr Gln Phe Leu Asn Gln His Leu AsnPro Ala 405 410 415 Ile Glu Ser Asp Asn Asn Phe Thr Asp Thr Thr Lys IlePhe His Asp 420 425 430 Ser Leu Phe Asn Ser Ala Thr Ala Glu Asn Ser MetPhe Leu Thr Lys 435 440 445 Ile Ala Pro Tyr Leu Gln Val Gly Phe Met ProGlu Ala Arg Ser Thr 450 455 460 Ile Ser Leu Ser Gly Pro Gly Ala Tyr AlaSer Ala Tyr Tyr Asp Phe 465 470 475 480 Ile Asn Leu Gln Glu Asn Thr IleGlu Lys Thr Leu Lys Ala Ser Asp 485 490 495 Leu Ile Glu Phe Lys Phe ProGlu Asn Asn Leu Ser Gln Leu Thr Glu 500 505 510 Gln Glu Ile Asn Ser LeuTrp Ser Phe Asp Gln Ala Ser Ala Lys Tyr 515 520 525 Gln Phe Glu Lys TyrVal Arg Asp Tyr Thr Gly Gly Ser Leu Ser Glu 530 535 540 Asp Asn Gly ValAsp Phe Asn Lys Asn Thr Ala Leu Asp Lys Asn Tyr 545 550 555 560 Leu LeuAsn Asn Lys Ile Pro Ser Asn Asn Val Glu Glu Ala Gly Ser 565 570 575 LysAsn Tyr Val His Tyr Ile Ile Gln Leu Gln Gly Asp Asp Ile Ser 580 585 590Tyr Glu Ala Thr Cys Asn Leu Phe Ser Lys Asn Pro Lys Asn Ser Ile 595 600605 Ile Ile Gln Arg Asn Met Asn Glu Ser Ala Lys Ser Tyr Phe Leu Ser 610615 620 Asp Asp Gly Glu Ser Ile Leu Glu Leu Asn Lys Tyr Arg Ile Pro Glu625 630 635 640 Arg Leu Lys Asn Lys Glu Lys Val Lys Val Thr Phe Ile GlyHis Gly 645 650 655 Lys Asp Glu Phe Asn Thr Ser Glu Phe Ala Arg Leu SerVal Asp Ser 660 665 670 Leu Ser Asn Glu Ile Ser Ser Phe Leu Asp Thr IleLys Leu Asp Ile 675 680 685 Ser Pro Lys Asn Val Glu Val Asn Leu Leu GlyCys Asn Met Phe Ser 690 695 700 Tyr Asp Phe Asn Val Glu Glu Thr Tyr ProGly Lys Leu Leu Leu Ser 705 710 715 720 Ile Met Asp Lys Ile Thr Ser ThrLeu Pro Asp Val Asn Lys Asn Ser 725 730 735 Ile Thr Ile Gly Ala Asn GlnTyr Glu Val Arg Ile Asn Ser Glu Gly 740 745 750 Arg Lys Glu Leu Leu AlaHis Ser Gly Lys Trp Ile Asn Lys Glu Glu 755 760 765 Ala Ile Met Ser AspLeu Ser Ser Lys Glu Tyr Ile Phe Phe Asp Ser 770 775 780 Ile Asp Asn LysLeu Lys Ala Lys Ser Lys Asn Ile Pro Gly Leu Ala 785 790 795 800 Ser IleSer Glu Asp Ile Lys Thr Leu Leu Leu Asp Ala Ser Val Ser 805 810 815 ProAsp Thr Lys Phe Ile Leu Asn Asn Leu Lys Leu Asn Ile Glu Ser 820 825 830Ser Ile Gly Asp Tyr Ile Tyr Tyr Glu Lys Leu Glu Pro Val Lys Asn 835 840845 Ile Ile His Asn Ser Ile Asp Asp Leu Ile Asp Glu Phe Asn Leu Leu 850855 860 Glu Asn Val Ser Asp Glu Leu Tyr Glu Leu Lys Lys Leu Asn Asn Leu865 870 875 880 Asp Glu Lys Tyr Leu Ile Ser Phe Glu Asp Ile Ser Lys AsnAsn Ser 885 890 895 Thr Tyr Ser Val Arg Phe Ile Asn Lys Ser Asn Gly GluSer Val Tyr 900 905 910 Val Glu Thr Glu Lys Glu Ile Phe Ser Lys Tyr SerGlu His Ile Thr 915 920 925 Lys Glu Ile Ser Thr Ile Lys Asn Ser Ile IleThr Asp Val Asn Gly 930 935 940 Asn Leu Leu Asp Asn Ile Gln Leu Asp HisThr Ser Gln Val Asn Thr 945 950 955 960 Leu Asn Ala Ala Phe Phe Ile GlnSer Leu Ile Asp Tyr Ser Ser Asn 965 970 975 Lys Asp Val Leu Asn Asp LeuSer Thr Ser Val Lys Val Gln Leu Tyr 980 985 990 Ala Gln Leu Phe Ser ThrGly Leu Asn Thr Ile Tyr Asp Ser Ile Gln 995 1000 1005 Leu Val Asn LeuIle Ser Asn Ala Val Asn Asp Thr Ile Asn Val Leu 1010 1015 1020 Pro ThrIle Thr Glu Gly Ile Pro Ile Val Ser Thr Ile Leu Asp Gly 1025 1030 10351040 Ile Asn Leu Gly Ala Ala Ile Lys Glu Leu Leu Asp Glu His Asp Pro1045 1050 1055 Leu Leu Lys Lys Glu Leu Glu Ala Lys Val Gly Val Leu AlaIle Asn 1060 1065 1070 Met Ser Leu Ser Ile Ala Ala Thr Val Ala Ser IleVal Gly Ile Gly 1075 1080 1085 Ala Glu Val Thr Ile Phe Leu Leu Pro IleAla Gly Ile Ser Ala Gly 1090 1095 1100 Ile Pro Ser Leu Val Asn Asn GluLeu Ile Leu His Asp Lys Ala Thr 1105 1110 1115 1120 Ser Val Val Asn TyrPhe Asn His Leu Ser Glu Ser Lys Lys Tyr Gly 1125 1130 1135 Pro Leu LysThr Glu Asp Asp Lys Ile Leu Val Pro Ile Asp Asp Leu 1140 1145 1150 ValIle Ser Glu Ile Asp Phe Asn Asn Asn Ser Ile Lys Leu Gly Thr 1155 11601165 Cys Asn Ile Leu Ala Met Glu Gly Gly Ser Gly His Thr Val Thr Gly1170 1175 1180 Asn Ile Asp His Phe Phe Ser Ser Pro Ser Ile Ser Ser HisIle Pro 1185 1190 1195 1200 Ser Leu Ser Ile Tyr Ser Ala Ile Gly Ile GluThr Glu Asn Leu Asp 1205 1210 1215 Phe Ser Lys Lys Ile Met Met Leu ProAsn Ala Pro Ser Arg Val Phe 1220 1225 1230 Trp Trp Glu Thr Gly Ala ValPro Gly Leu Arg Ser Leu Glu Asn Asp 1235 1240 1245 Gly Thr Arg Leu LeuAsp Ser Ile Arg Asp Leu Tyr Pro Gly Lys Phe 1250 1255 1260 Tyr Trp ArgPhe Tyr Ala Phe Phe Asp Tyr Ala Ile Thr Thr Leu Lys 1265 1270 1275 1280Pro Val Tyr Glu Asp Thr Asn Ile Lys Ile Lys Leu Asp Lys Asp Thr 12851290 1295 Arg Asn Phe Ile Met Pro Thr Ile Thr Thr Asn Glu Ile Arg AsnLys 1300 1305 1310 Leu Ser Tyr Ser Phe Asp Gly Ala Gly Gly Thr Tyr SerLeu Leu Leu 1315 1320 1325 Ser Ser Tyr Pro Ile Ser Thr Asn Ile Asn LeuSer Lys Asp Asp Leu 1330 1335 1340 Trp Ile Phe Asn Ile Asp Asn Glu ValArg Glu Ile Ser Ile Glu Asn 1345 1350 1355 1360 Gly Thr Ile Lys Lys GlyLys Leu Ile Lys Asp Val Leu Ser Lys Ile 1365 1370 1375 Asp Ile Asn LysAsn Lys Leu Ile Ile Gly Asn Gln Thr Ile Asp Phe 1380 1385 1390 Ser GlyAsp Ile Asp Asn Lys Asp Arg Tyr Ile Phe Leu Thr Cys Glu 1395 1400 1405Leu Asp Asp Lys Ile Ser Leu Ile Ile Glu Ile Asn Leu Val Ala Lys 14101415 1420 Ser Tyr Ser Leu Leu Leu Ser Gly Asp Lys Asn Tyr Leu Ile SerAsn 1425 1430 1435 1440 Leu Ser Asn Thr Ile Glu Lys Ile Asn Thr Leu GlyLeu Asp Ser Lys 1445 1450 1455 Asn Ile Ala Tyr Asn Tyr Thr Asp Glu SerAsn Asn Lys Tyr Phe Gly 1460 1465 1470 Ala Ile Ser Lys Thr Ser Gln LysSer Ile Ile His Tyr Lys Lys Asp 1475 1480 1485 Ser Lys Asn Ile Leu GluPhe Tyr Asn Asp Ser Thr Leu Glu Phe Asn 1490 1495 1500 Ser Lys Asp PheIle Ala Glu Asp Ile Asn Val Phe Met Lys Asp Asp 1505 1510 1515 1520 IleAsn Thr Ile Thr Gly Lys Tyr Tyr Val Asp Asn Asn Thr Asp Lys 1525 15301535 Ser Ile Asp Phe Ser Ile Ser Leu Val Ser Lys Asn Gln Val Lys Val1540 1545 1550 Asn Gly Leu Tyr Leu Asn Glu Ser Val Tyr Ser Ser Tyr LeuAsp Phe 1555 1560 1565 Val Lys Asn Ser Asp Gly His His Asn Thr Ser AsnPhe Met Asn Leu 1570 1575 1580 Phe Leu Asp Asn Ile Ser Phe Trp Lys LeuPhe Gly Phe Glu Asn Ile 1585 1590 1595 1600 Asn Phe Val Ile Asp Lys TyrPhe Thr Leu Val Gly Lys Thr Asn Leu 1605 1610 1615 Gly Tyr Val Glu PheIle Cys Asp Asn Asn Lys Asn Ile Asp Ile Tyr 1620 1625 1630 Phe Gly GluTrp Lys Thr Ser Ser Ser Lys Ser Thr Ile Phe Ser Gly 1635 1640 1645 AsnGly Arg Asn Val Val Val Glu Pro Ile Tyr Asn Pro Asp Thr Gly 1650 16551660 Glu Asp Ile Ser Thr Ser Leu Asp Phe Ser Tyr Glu Pro Leu Tyr Gly1665 1670 1675 1680 Ile Asp Arg Tyr Ile Asn Lys Val Leu Ile Ala Pro AspLeu Tyr Thr 1685 1690 1695 Ser Leu Ile Asn Ile Asn Thr Asn Tyr Tyr SerAsn Glu Tyr Tyr Pro 1700 1705 1710 Glu Ile Ile Val Leu Asn Pro Asn ThrPhe His Lys Lys Val Asn Ile 1715 1720 1725 Asn Leu Asp Ser Ser Ser PheGlu Tyr Lys Trp Ser Thr Glu Gly Ser 1730 1735 1740 Asp Phe Ile Leu ValArg Tyr Leu Glu Glu Ser Asn Lys Lys Ile Leu 1745 1750 1755 1760 Gln LysIle Arg Ile Lys Gly Ile Leu Ser Asn Thr Gln Ser Phe Asn 1765 1770 1775Lys Met Ser Ile Asp Phe Lys Asp Ile Lys Lys Leu Ser Leu Gly Tyr 17801785 1790 Ile Met Ser Asn Phe Lys Ser Phe Asn Ser Glu Asn Glu Leu AspArg 1795 1800 1805 Asp His Leu Gly Phe Lys Ile Ile Asp Asn Lys Thr TyrTyr Tyr Asp 1810 1815 1820 Glu Asp Ser Lys Leu Val Lys Gly Leu Ile AsnIle Asn Asn Ser Leu 1825 1830 1835 1840 Phe Tyr Phe Asp Pro Ile Glu PheAsn Leu Val Thr Gly Trp Gln Thr 1845 1850 1855 Ile Asn Gly Lys Lys TyrTyr Phe Asp Ile Asn Thr Gly Ala Ala Leu 1860 1865 1870 Thr Ser Tyr LysIle Ile Asn Gly Lys His Phe Tyr Phe Asn Asn Asp 1875 1880 1885 Gly ValMet Gln Leu Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr 1890 1895 1900Phe Ala Pro Ala Asn Thr Gln Asn Asn Asn Ile Glu Gly Gln Ala Ile 19051910 1915 1920 Val Tyr Gln Ser Lys Phe Leu Thr Leu Asn Gly Lys Lys TyrTyr Phe 1925 1930 1935 Asp Asn Asn Ser Lys Ala Val Thr Gly Trp Arg IleIle Asn Asn Glu 1940 1945 1950 Lys Tyr Tyr Phe Asn Pro Asn Asn Ala IleAla Ala Val Gly Leu Gln 1955 1960 1965 Val Ile Asp Asn Asn Lys Tyr TyrPhe Asn Pro Asp Thr Ala Ile Ile 1970 1975 1980 Ser Lys Gly Trp Gln ThrVal Asn Gly Ser Arg Tyr Tyr Phe Asp Thr 1985 1990 1995 2000 Asp Thr AlaIle Ala Phe Asn Gly Tyr Lys Thr Ile Asp Gly Lys His 2005 2010 2015 PheTyr Phe Asp Ser Asp Cys Val Val Lys Ile Gly Val Phe Ser Thr 2020 20252030 Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Tyr Asn Asn Asn2035 2040 2045 Ile Glu Gly Gln Ala Ile Val Tyr Gln Ser Lys Phe Leu ThrLeu Asn 2050 2055 2060 Gly Lys Lys Tyr Tyr Phe Asp Asn Asn Ser Lys AlaVal Thr Gly Leu 2065 2070 2075 2080 Gln Thr Ile Asp Ser Lys Lys Tyr TyrPhe Asn Thr Asn Thr Ala Glu 2085 2090 2095 Ala Ala Thr Gly Trp Gln ThrIle Asp Gly Lys Lys Tyr Tyr Phe Asn 2100 2105 2110 Thr Asn Thr Ala GluAla Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys 2115 2120 2125 Lys Tyr TyrPhe Asn Thr Asn Thr Ala Ile Ala Ser Thr Gly Tyr Thr 2130 2135 2140 IleIle Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile Met Gln 2145 21502155 2160 Ile Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala ProAla 2165 2170 2175 Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile LeuTyr Gln Asn 2180 2185 2190 Glu Phe Leu Thr Leu Asn Gly Lys Lys Tyr TyrPhe Gly Ser Asp Ser 2195 2200 2205 Lys Ala Val Thr Gly Trp Arg Ile IleAsn Asn Lys Lys Tyr Tyr Phe 2210 2215 2220 Asn Pro Asn Asn Ala Ile AlaAla Ile His Leu Cys Thr Ile Asn Asn 2225 2230 2235 2240 Asp Lys Tyr TyrPhe Ser Tyr Asp Gly Ile Leu Gln Asn Gly Tyr Ile 2245 2250 2255 Thr IleGlu Arg Asn Asn Phe Tyr Phe Asp Ala Asn Asn Glu Ser Lys 2260 2265 2270Met Val Thr Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala 22752280 2285 Pro Ala Asn Thr His Asn Asn Asn Ile Glu Gly Gln Ala Ile ValTyr 2290 2295 2300 Gln Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr TyrPhe Asp Asn 2305 2310 2315 2320 Asp Ser Lys Ala Val Thr Gly Trp Gln ThrIle Asp Gly Lys Lys Tyr 2325 2330 2335 Tyr Phe Asn Leu Asn Thr Ala GluAla Ala Thr Gly Trp Gln Thr Ile 2340 2345 2350 Asp Gly Lys Lys Tyr TyrPhe Asn Leu Asn Thr Ala Glu Ala Ala Thr 2355 2360 2365 Gly Trp Gln ThrIle Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr 2370 2375 2380 Phe IleAla Ser Thr Gly Tyr Thr Ser Ile Asn Gly Lys His Phe Tyr 2385 2390 23952400 Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asn2405 2410 2415 Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn AsnIle Glu 2420 2425 2430 Gly Gln Ala Ile Leu Tyr Gln Asn Lys Phe Leu ThrLeu Asn Gly Lys 2435 2440 2445 Lys Tyr Tyr Phe Gly Ser Asp Ser Lys AlaVal Thr Gly Leu Arg Thr 2450 2455 2460 Ile Asp Gly Lys Lys Tyr Tyr PheAsn Thr Asn Thr Ala Val Ala Val 2465 2470 2475 2480 Thr Gly Trp Gln ThrIle Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn 2485 2490 2495 Thr Ser IleAla Ser Thr Gly Tyr Thr Ile Ile Ser Gly Lys His Phe 2500 2505 2510 TyrPhe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro 2515 25202525 Asp Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile2530 2535 2540 Glu Gly Gln Ala Ile Arg Tyr Gln Asn Arg Phe Leu Tyr LeuHis Asp 2545 2550 2555 2560 Asn Ile Tyr Tyr Phe Gly Asn Asn Ser Lys AlaAla Thr Gly Trp Val 2565 2570 2575 Thr Ile Asp Gly Asn Arg Tyr Tyr PheGlu Pro Asn Thr Ala Met Gly 2580 2585 2590 Ala Asn Gly Tyr Lys Thr IleAsp Asn Lys Asn Phe Tyr Phe Arg Asn 2595 2600 2605 Gly Leu Pro Gln IleGly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr 2610 2615 2620 Phe Ala ProAla Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile 2625 2630 2635 2640Arg Tyr Gln Asn Arg Phe Leu His Leu Leu Gly Lys Ile Tyr Tyr Phe 26452650 2655 Gly Asn Asn Ser Lys Ala Val Thr Gly Trp Gln Thr Ile Asn GlyLys 2660 2665 2670 Val Tyr Tyr Phe Met Pro Asp Thr Ala Met Ala Ala AlaGly Gly Leu 2675 2680 2685 Phe Glu Ile Asp Gly Val Ile Tyr Phe Phe GlyVal Asp Gly Val Lys 2690 2695 2700 Ala Pro Gly Ile Tyr Gly 2705 2710 811amino acids amino acid unknown unknown protein 7 Ser Tyr Lys Ile Ile AsnGly Lys His Phe Tyr Phe Asn Asn Asp Gly 1 5 10 15 Val Met Gln Leu GlyVal Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe 20 25 30 Ala Pro Ala Asn ThrGln Asn Asn Asn Ile Glu Gly Gln Ala Ile Val 35 40 45 Tyr Gln Ser Lys PheLeu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp 50 55 60 Asn Asn Ser Lys AlaVal Thr Gly Trp Arg Ile Ile Asn Asn Glu Lys 65 70 75 80 Tyr Tyr Phe AsnPro Asn Asn Ala Ile Ala Ala Val Gly Leu Gln Val 85 90 95 Ile Asp Asn AsnLys Tyr Tyr Phe Asn Pro Asp Thr Ala Ile Ile Ser 100 105 110 Lys Gly TrpGln Thr Val Asn Gly Ser Arg Tyr Tyr Phe Asp Thr Asp 115 120 125 Thr AlaIle Ala Phe Asn Gly Tyr Lys Thr Ile Asp Gly Lys His Phe 130 135 140 TyrPhe Asp Ser Asp Cys Val Val Lys Ile Gly Val Phe Ser Thr Ser 145 150 155160 Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Tyr Asn Asn Asn Ile 165170 175 Glu Gly Gln Ala Ile Val Tyr Gln Ser Lys Phe Leu Thr Leu Asn Gly180 185 190 Lys Lys Tyr Tyr Phe Asp Asn Asn Ser Lys Ala Val Thr Gly LeuGln 195 200 205 Thr Ile Asp Ser Lys Lys Tyr Tyr Phe Asn Thr Asn Thr AlaGlu Ala 210 215 220 Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr TyrPhe Asn Thr 225 230 235 240 Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln ThrIle Asp Gly Lys Lys 245 250 255 Tyr Tyr Phe Asn Thr Asn Thr Ala Ile AlaSer Thr Gly Tyr Thr Ile 260 265 270 Ile Asn Gly Lys His Phe Tyr Phe AsnThr Asp Gly Ile Met Gln Ile 275 280 285 Gly Val Phe Lys Gly Pro Asn GlyPhe Glu Tyr Phe Ala Pro Ala Asn 290 295 300 Thr Asp Ala Asn Asn Ile GluGly Gln Ala Ile Leu Tyr Gln Asn Glu 305 310 315 320 Phe Leu Thr Leu AsnGly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys 325 330 335 Ala Val Thr GlyTrp Arg Ile Ile Asn Asn Lys Lys Tyr Tyr Phe Asn 340 345 350 Pro Asn AsnAla Ile Ala Ala Ile His Leu Cys Thr Ile Asn Asn Asp 355 360 365 Lys TyrTyr Phe Ser Tyr Asp Gly Ile Leu Gln Asn Gly Tyr Ile Thr 370 375 380 IleGlu Arg Asn Asn Phe Tyr Phe Asp Ala Asn Asn Glu Ser Lys Met 385 390 395400 Val Thr Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro 405410 415 Ala Asn Thr His Asn Asn Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln420 425 430 Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp AsnAsp 435 440 445 Ser Lys Ala Val Thr Gly Trp Gln Thr Ile Asp Gly Lys LysTyr Tyr 450 455 460 Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp GlnThr Ile Asp 465 470 475 480 Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr AlaGlu Ala Ala Thr Gly 485 490 495 Trp Gln Thr Ile Asp Gly Lys Lys Tyr TyrPhe Asn Thr Asn Thr Phe 500 505 510 Ile Ala Ser Thr Gly Tyr Thr Ser IleAsn Gly Lys His Phe Tyr Phe 515 520 525 Asn Thr Asp Gly Ile Met Gln IleGly Val Phe Lys Gly Pro Asn Gly 530 535 540 Phe Glu Tyr Phe Ala Pro AlaAsn Thr Asp Ala Asn Asn Ile Glu Gly 545 550 555 560 Gln Ala Ile Leu TyrGln Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys 565 570 575 Tyr Tyr Phe GlySer Asp Ser Lys Ala Val Thr Gly Leu Arg Thr Ile 580 585 590 Asp Gly LysLys Tyr Tyr Phe Asn Thr Asn Thr Ala Val Ala Val Thr 595 600 605 Gly TrpGln Thr Ile Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr 610 615 620 SerIle Ala Ser Thr Gly Tyr Thr Ile Ile Ser Gly Lys His Phe Tyr 625 630 635640 Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asp 645650 655 Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu660 665 670 Gly Gln Ala Ile Arg Tyr Gln Asn Arg Phe Leu Tyr Leu His AspAsn 675 680 685 Ile Tyr Tyr Phe Gly Asn Asn Ser Lys Ala Ala Thr Gly TrpVal Thr 690 695 700 Ile Asp Gly Asn Arg Tyr Tyr Phe Glu Pro Asn Thr AlaMet Gly Ala 705 710 715 720 Asn Gly Tyr Lys Thr Ile Asp Asn Lys Asn PheTyr Phe Arg Asn Gly 725 730 735 Leu Pro Gln Ile Gly Val Phe Lys Gly SerAsn Gly Phe Glu Tyr Phe 740 745 750 Ala Pro Ala Asn Thr Asp Ala Asn AsnIle Glu Gly Gln Ala Ile Arg 755 760 765 Tyr Gln Asn Arg Phe Leu His LeuLeu Gly Lys Ile Tyr Tyr Phe Gly 770 775 780 Asn Asn Ser Lys Ala Val ThrGly Trp Gln Thr Ile Asn Gly Lys Val 785 790 795 800 Tyr Tyr Phe Met ProAsp Thr Ala Met Ala Ala 805 810 91 amino acids amino acid unknownunknown protein 8 Ser Tyr Lys Ile Ile Asn Gly Lys His Phe Tyr Phe AsnAsn Asp Gly 1 5 10 15 Val Met Gln Leu Gly Val Phe Lys Gly Pro Asp GlyPhe Glu Tyr Phe 20 25 30 Ala Pro Ala Asn Thr Gln Asn Asn Asn Ile Glu GlyGln Ala Ile Val 35 40 45 Tyr Gln Ser Lys Phe Leu Thr Leu Asn Gly Lys LysTyr Tyr Phe Asp 50 55 60 Asn Asn Ser Lys Ala Val Thr Gly Trp Arg Ile IleAsn Asn Glu Lys 65 70 75 80 Tyr Tyr Phe Asn Pro Asn Asn Ala Ile Ala Ala85 90 7101 base pairs nucleic acid single linear DNA (genomic) CDS1..7098 9 ATG AGT TTA GTT AAT AGA AAA CAG TTA GAA AAA ATG GCA AAT GTAAGA 48 Met Ser Leu Val Asn Arg Lys Gln Leu Glu Lys Met Ala Asn Val Arg 15 10 15 TTT CGT ACT CAA GAA GAT GAA TAT GTT GCA ATA TTG GAT GCT TTA GAA96 Phe Arg Thr Gln Glu Asp Glu Tyr Val Ala Ile Leu Asp Ala Leu Glu 20 2530 GAA TAT CAT AAT ATG TCA GAG AAT ACT GTA GTC GAA AAA TAT TTA AAA 144Glu Tyr His Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu Lys 35 40 45TTA AAA GAT ATA AAT AGT TTA ACA GAT ATT TAT ATA GAT ACA TAT AAA 192 LeuLys Asp Ile Asn Ser Leu Thr Asp Ile Tyr Ile Asp Thr Tyr Lys 50 55 60 AAATCT GGT AGA AAT AAA GCC TTA AAA AAA TTT AAG GAA TAT CTA GTT 240 Lys SerGly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu Tyr Leu Val 65 70 75 80 ACAGAA GTA TTA GAG CTA AAG AAT AAT AAT TTA ACT CCA GTT GAG AAA 288 Thr GluVal Leu Glu Leu Lys Asn Asn Asn Leu Thr Pro Val Glu Lys 85 90 95 AAT TTACAT TTT GTT TGG ATT GGA GGT CAA ATA AAT GAC ACT GCT ATT 336 Asn Leu HisPhe Val Trp Ile Gly Gly Gln Ile Asn Asp Thr Ala Ile 100 105 110 AAT TATATA AAT CAA TGG AAA GAT GTA AAT AGT GAT TAT AAT GTT AAT 384 Asn Tyr IleAsn Gln Trp Lys Asp Val Asn Ser Asp Tyr Asn Val Asn 115 120 125 GTT TTTTAT GAT AGT AAT GCA TTT TTG ATA AAC ACA TTG AAA AAA ACT 432 Val Phe TyrAsp Ser Asn Ala Phe Leu Ile Asn Thr Leu Lys Lys Thr 130 135 140 GTA GTAGAA TCA GCA ATA AAT GAT ACA CTT GAA TCA TTT AGA GAA AAC 480 Val Val GluSer Ala Ile Asn Asp Thr Leu Glu Ser Phe Arg Glu Asn 145 150 155 160 TTAAAT GAC CCT AGA TTT GAC TAT AAT AAA TTC TTC AGA AAA CGT ATG 528 Leu AsnAsp Pro Arg Phe Asp Tyr Asn Lys Phe Phe Arg Lys Arg Met 165 170 175 GAAATA ATT TAT GAT AAA CAG AAA AAT TTC ATA AAC TAC TAT AAA GCT 576 Glu IleIle Tyr Asp Lys Gln Lys Asn Phe Ile Asn Tyr Tyr Lys Ala 180 185 190 CAAAGA GAA GAA AAT CCT GAA CTT ATA ATT GAT GAT ATT GTA AAG ACA 624 Gln ArgGlu Glu Asn Pro Glu Leu Ile Ile Asp Asp Ile Val Lys Thr 195 200 205 TATCTT TCA AAT GAG TAT TCA AAG GAG ATA GAT GAA CTT AAT ACC TAT 672 Tyr LeuSer Asn Glu Tyr Ser Lys Glu Ile Asp Glu Leu Asn Thr Tyr 210 215 220 ATTGAA GAA TCC TTA AAT AAA ATT ACA CAG AAT AGT GGA AAT GAT GTT 720 Ile GluGlu Ser Leu Asn Lys Ile Thr Gln Asn Ser Gly Asn Asp Val 225 230 235 240AGA AAC TTT GAA GAA TTT AAA AAT GGA GAG TCA TTC AAC TTA TAT GAA 768 ArgAsn Phe Glu Glu Phe Lys Asn Gly Glu Ser Phe Asn Leu Tyr Glu 245 250 255CAA GAG TTG GTA GAA AGG TGG AAT TTA GCT GCT GCT TCT GAC ATA TTA 816 GlnGlu Leu Val Glu Arg Trp Asn Leu Ala Ala Ala Ser Asp Ile Leu 260 265 270AGA ATA TCT GCA TTA AAA GAA ATT GGT GGT ATG TAT TTA GAT GTT GAT 864 ArgIle Ser Ala Leu Lys Glu Ile Gly Gly Met Tyr Leu Asp Val Asp 275 280 285ATG TTA CCA GGA ATA CAA CCA GAC TTA TTT GAG TCT ATA GAG AAA CCT 912 MetLeu Pro Gly Ile Gln Pro Asp Leu Phe Glu Ser Ile Glu Lys Pro 290 295 300AGT TCA GTA ACA GTG GAT TTT TGG GAA ATG ACA AAG TTA GAA GCT ATA 960 SerSer Val Thr Val Asp Phe Trp Glu Met Thr Lys Leu Glu Ala Ile 305 310 315320 ATG AAA TAC AAA GAA TAT ATA CCA GAA TAT ACC TCA GAA CAT TTT GAC 1008Met Lys Tyr Lys Glu Tyr Ile Pro Glu Tyr Thr Ser Glu His Phe Asp 325 330335 ATG TTA GAC GAA GAA GTT CAA AGT AGT TTT GAA TCT GTT CTA GCT TCT 1056Met Leu Asp Glu Glu Val Gln Ser Ser Phe Glu Ser Val Leu Ala Ser 340 345350 AAG TCA GAT AAA TCA GAA ATA TTC TCA TCA CTT GGT GAT ATG GAG GCA 1104Lys Ser Asp Lys Ser Glu Ile Phe Ser Ser Leu Gly Asp Met Glu Ala 355 360365 TCA CCA CTA GAA GTT AAA ATT GCA TTT AAT AGT AAG GGT ATT ATA AAT 1152Ser Pro Leu Glu Val Lys Ile Ala Phe Asn Ser Lys Gly Ile Ile Asn 370 375380 CAA GGG CTA ATT TCT GTG AAA GAC TCA TAT TGT AGC AAT TTA ATA GTA 1200Gln Gly Leu Ile Ser Val Lys Asp Ser Tyr Cys Ser Asn Leu Ile Val 385 390395 400 AAA CAA ATC GAG AAT AGA TAT AAA ATA TTG AAT AAT AGT TTA AAT CCA1248 Lys Gln Ile Glu Asn Arg Tyr Lys Ile Leu Asn Asn Ser Leu Asn Pro 405410 415 GCT ATT AGC GAG GAT AAT GAT TTT AAT ACT ACA ACG AAT ACC TTT ATT1296 Ala Ile Ser Glu Asp Asn Asp Phe Asn Thr Thr Thr Asn Thr Phe Ile 420425 430 GAT AGT ATA ATG GCT GAA GCT AAT GCA GAT AAT GGT AGA TTT ATG ATG1344 Asp Ser Ile Met Ala Glu Ala Asn Ala Asp Asn Gly Arg Phe Met Met 435440 445 GAA CTA GGA AAG TAT TTA AGA GTT GGT TTC TTC CCA GAT GTT AAA ACT1392 Glu Leu Gly Lys Tyr Leu Arg Val Gly Phe Phe Pro Asp Val Lys Thr 450455 460 ACT ATT AAC TTA AGT GGC CCT GAA GCA TAT GCG GCA GCT TAT CAA GAT1440 Thr Ile Asn Leu Ser Gly Pro Glu Ala Tyr Ala Ala Ala Tyr Gln Asp 465470 475 480 TTA TTA ATG TTT AAA GAA GGC AGT ATG AAT ATC CAT TTG ATA GAAGCT 1488 Leu Leu Met Phe Lys Glu Gly Ser Met Asn Ile His Leu Ile Glu Ala485 490 495 GAT TTA AGA AAC TTT GAA ATC TCT AAA ACT AAT ATT TCT CAA TCAACT 1536 Asp Leu Arg Asn Phe Glu Ile Ser Lys Thr Asn Ile Ser Gln Ser Thr500 505 510 GAA CAA GAA ATG GCT AGC TTA TGG TCA TTT GAC GAT GCA AGA GCTAAA 1584 Glu Gln Glu Met Ala Ser Leu Trp Ser Phe Asp Asp Ala Arg Ala Lys515 520 525 GCT CAA TTT GAA GAA TAT AAA AGG AAT TAT TTT GAA GGT TCT CTTGGT 1632 Ala Gln Phe Glu Glu Tyr Lys Arg Asn Tyr Phe Glu Gly Ser Leu Gly530 535 540 GAA GAT GAT AAT CTT GAT TTT TCT CAA AAT ATA GTA GTT GAC AAGGAG 1680 Glu Asp Asp Asn Leu Asp Phe Ser Gln Asn Ile Val Val Asp Lys Glu545 550 555 560 TAT CTT TTA GAA AAA ATA TCT TCA TTA GCA AGA AGT TCA GAGAGA GGA 1728 Tyr Leu Leu Glu Lys Ile Ser Ser Leu Ala Arg Ser Ser Glu ArgGly 565 570 575 TAT ATA CAC TAT ATT GTT CAG TTA CAA GGA GAT AAA ATT AGTTAT GAA 1776 Tyr Ile His Tyr Ile Val Gln Leu Gln Gly Asp Lys Ile Ser TyrGlu 580 585 590 GCA GCA TGT AAC TTA TTT GCA AAG ACT CCT TAT GAT AGT GTACTG TTT 1824 Ala Ala Cys Asn Leu Phe Ala Lys Thr Pro Tyr Asp Ser Val LeuPhe 595 600 605 CAG AAA AAT ATA GAA GAT TCA GAA ATT GCA TAT TAT TAT AATCCT GGA 1872 Gln Lys Asn Ile Glu Asp Ser Glu Ile Ala Tyr Tyr Tyr Asn ProGly 610 615 620 GAT GGT GAA ATA CAA GAA ATA GAC AAG TAT AAA ATT CCA AGTATA ATT 1920 Asp Gly Glu Ile Gln Glu Ile Asp Lys Tyr Lys Ile Pro Ser IleIle 625 630 635 640 TCT GAT AGA CCT AAG ATT AAA TTA ACA TTT ATT GGT CATGGT AAA GAT 1968 Ser Asp Arg Pro Lys Ile Lys Leu Thr Phe Ile Gly His GlyLys Asp 645 650 655 GAA TTT AAT ACT GAT ATA TTT GCA GGT TTT GAT GTA GATTCA TTA TCC 2016 Glu Phe Asn Thr Asp Ile Phe Ala Gly Phe Asp Val Asp SerLeu Ser 660 665 670 ACA GAA ATA GAA GCA GCA ATA GAT TTA GCT AAA GAG GATATT TCT CCT 2064 Thr Glu Ile Glu Ala Ala Ile Asp Leu Ala Lys Glu Asp IleSer Pro 675 680 685 AAG TCA ATA GAA ATA AAT TTA TTA GGA TGT AAT ATG TTTAGC TAC TCT 2112 Lys Ser Ile Glu Ile Asn Leu Leu Gly Cys Asn Met Phe SerTyr Ser 690 695 700 ATC AAC GTA GAG GAG ACT TAT CCT GGA AAA TTA TTA CTTAAA GTT AAA 2160 Ile Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu LysVal Lys 705 710 715 720 GAT AAA ATA TCA GAA TTA ATG CCA TCT ATA AGT CAAGAC TCT ATT ATA 2208 Asp Lys Ile Ser Glu Leu Met Pro Ser Ile Ser Gln AspSer Ile Ile 725 730 735 GTA AGT GCA AAT CAA TAT GAA GTT AGA ATA AAT AGTGAA GGA AGA AGA 2256 Val Ser Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser GluGly Arg Arg 740 745 750 GAA TTA TTG GAT CAT TCT GGT GAA TGG ATA AAT AAAGAA GAA AGT ATT 2304 Glu Leu Leu Asp His Ser Gly Glu Trp Ile Asn Lys GluGlu Ser Ile 755 760 765 ATA AAG GAT ATT TCA TCA AAA GAA TAT ATA TCA TTTAAT CCT AAA GAA 2352 Ile Lys Asp Ile Ser Ser Lys Glu Tyr Ile Ser Phe AsnPro Lys Glu 770 775 780 AAT AAA ATT ACA GTA AAA TCT AAA AAT TTA CCT GAGCTA TCT ACA TTA 2400 Asn Lys Ile Thr Val Lys Ser Lys Asn Leu Pro Glu LeuSer Thr Leu 785 790 795 800 TTA CAA GAA ATT AGA AAT AAT TCT AAT TCA AGTGAT ATT GAA CTA GAA 2448 Leu Gln Glu Ile Arg Asn Asn Ser Asn Ser Ser AspIle Glu Leu Glu 805 810 815 GAA AAA GTA ATG TTA ACA GAA TGT GAG ATA AATGTT ATT TCA AAT ATA 2496 Glu Lys Val Met Leu Thr Glu Cys Glu Ile Asn ValIle Ser Asn Ile 820 825 830 GAT ACG CAA ATT GTT GAG GAA AGG ATT GAA GAAGCT AAG AAT TTA ACT 2544 Asp Thr Gln Ile Val Glu Glu Arg Ile Glu Glu AlaLys Asn Leu Thr 835 840 845 TCT GAC TCT ATT AAT TAT ATA AAA GAT GAA TTTAAA CTA ATA GAA TCT 2592 Ser Asp Ser Ile Asn Tyr Ile Lys Asp Glu Phe LysLeu Ile Glu Ser 850 855 860 ATT TCT GAT GCA CTA TGT GAC TTA AAA CAA CAGAAT GAA TTA GAA GAT 2640 Ile Ser Asp Ala Leu Cys Asp Leu Lys Gln Gln AsnGlu Leu Glu Asp 865 870 875 880 TCT CAT TTT ATA TCT TTT GAG GAC ATA TCAGAG ACT GAT GAG GGA TTT 2688 Ser His Phe Ile Ser Phe Glu Asp Ile Ser GluThr Asp Glu Gly Phe 885 890 895 AGT ATA AGA TTT ATT AAT AAA GAA ACT GGAGAA TCT ATA TTT GTA GAA 2736 Ser Ile Arg Phe Ile Asn Lys Glu Thr Gly GluSer Ile Phe Val Glu 900 905 910 ACT GAA AAA ACA ATA TTC TCT GAA TAT GCTAAT CAT ATA ACT GAA GAG 2784 Thr Glu Lys Thr Ile Phe Ser Glu Tyr Ala AsnHis Ile Thr Glu Glu 915 920 925 ATT TCT AAG ATA AAA GGT ACT ATA TTT GATACT GTA AAT GGT AAG TTA 2832 Ile Ser Lys Ile Lys Gly Thr Ile Phe Asp ThrVal Asn Gly Lys Leu 930 935 940 GTA AAA AAA GTA AAT TTA GAT ACT ACA CACGAA GTA AAT ACT TTA AAT 2880 Val Lys Lys Val Asn Leu Asp Thr Thr His GluVal Asn Thr Leu Asn 945 950 955 960 GCT GCA TTT TTT ATA CAA TCA TTA ATAGAA TAT AAT AGT TCT AAA GAA 2928 Ala Ala Phe Phe Ile Gln Ser Leu Ile GluTyr Asn Ser Ser Lys Glu 965 970 975 TCT CTT AGT AAT TTA AGT GTA GCA ATGAAA GTC CAA GTT TAC GCT CAA 2976 Ser Leu Ser Asn Leu Ser Val Ala Met LysVal Gln Val Tyr Ala Gln 980 985 990 TTA TTT AGT ACT GGT TTA AAT ACT ATTACA GAT GCA GCC AAA GTT GTT 3024 Leu Phe Ser Thr Gly Leu Asn Thr Ile ThrAsp Ala Ala Lys Val Val 995 1000 1005 GAA TTA GTA TCA ACT GCA TTA GATGAA ACT ATA GAC TTA CTT CCT ACA 3072 Glu Leu Val Ser Thr Ala Leu Asp GluThr Ile Asp Leu Leu Pro Thr 1010 1015 1020 TTA TCT GAA GGA TTA CCT ATAATT GCA ACT ATT ATA GAT GGT GTA AGT 3120 Leu Ser Glu Gly Leu Pro Ile IleAla Thr Ile Ile Asp Gly Val Ser 1025 1030 1035 1040 TTA GGT GCA GCA ATCAAA GAG CTA AGT GAA ACG AGT GAC CCA TTA TTA 3168 Leu Gly Ala Ala Ile LysGlu Leu Ser Glu Thr Ser Asp Pro Leu Leu 1045 1050 1055 AGA CAA GAA ATAGAA GCT AAG ATA GGT ATA ATG GCA GTA AAT TTA ACA 3216 Arg Gln Glu Ile GluAla Lys Ile Gly Ile Met Ala Val Asn Leu Thr 1060 1065 1070 ACA GCT ACAACT GCA ATC ATT ACT TCA TCT TTG GGG ATA GCT AGT GGA 3264 Thr Ala Thr ThrAla Ile Ile Thr Ser Ser Leu Gly Ile Ala Ser Gly 1075 1080 1085 TTT AGTATA CTT TTA GTT CCT TTA GCA GGA ATT TCA GCA GGT ATA CCA 3312 Phe Ser IleLeu Leu Val Pro Leu Ala Gly Ile Ser Ala Gly Ile Pro 1090 1095 1100 AGCTTA GTA AAC AAT GAA CTT GTA CTT CGA GAT AAG GCA ACA AAG GTT 3360 Ser LeuVal Asn Asn Glu Leu Val Leu Arg Asp Lys Ala Thr Lys Val 1105 1110 11151120 GTA GAT TAT TTT AAA CAT GTT TCA TTA GTT GAA ACT GAA GGA GTA TTT3408 Val Asp Tyr Phe Lys His Val Ser Leu Val Glu Thr Glu Gly Val Phe1125 1130 1135 ACT TTA TTA GAT GAT AAA ATA ATG ATG CCA CAA GAT GAT TTAGTG ATA 3456 Thr Leu Leu Asp Asp Lys Ile Met Met Pro Gln Asp Asp Leu ValIle 1140 1145 1150 TCA GAA ATA GAT TTT AAT AAT AAT TCA ATA GTT TTA GGTAAA TGT GAA 3504 Ser Glu Ile Asp Phe Asn Asn Asn Ser Ile Val Leu Gly LysCys Glu 1155 1160 1165 ATC TGG AGA ATG GAA GGT GGT TCA GGT CAT ACT GTAACT GAT GAT ATA 3552 Ile Trp Arg Met Glu Gly Gly Ser Gly His Thr Val ThrAsp Asp Ile 1170 1175 1180 GAT CAC TTC TTT TCA GCA CCA TCA ATA ACA TATAGA GAG CCA CAC TTA 3600 Asp His Phe Phe Ser Ala Pro Ser Ile Thr Tyr ArgGlu Pro His Leu 1185 1190 1195 1200 TCT ATA TAT GAC GTA TTG GAA GTA CAAAAA GAA GAA CTT GAT TTG TCA 3648 Ser Ile Tyr Asp Val Leu Glu Val Gln LysGlu Glu Leu Asp Leu Ser 1205 1210 1215 AAA GAT TTA ATG GTA TTA CCT AATGCT CCA AAT AGA GTA TTT GCT TGG 3696 Lys Asp Leu Met Val Leu Pro Asn AlaPro Asn Arg Val Phe Ala Trp 1220 1225 1230 GAA ACA GGA TGG ACA CCA GGTTTA AGA AGC TTA GAA AAT GAT GGC ACA 3744 Glu Thr Gly Trp Thr Pro Gly LeuArg Ser Leu Glu Asn Asp Gly Thr 1235 1240 1245 AAA CTG TTA GAC CGT ATAAGA GAT AAC TAT GAA GGT GAG TTT TAT TGG 3792 Lys Leu Leu Asp Arg Ile ArgAsp Asn Tyr Glu Gly Glu Phe Tyr Trp 1250 1255 1260 AGA TAT TTT GCT TTTATA GCT GAT GCT TTA ATA ACA ACA TTA AAA CCA 3840 Arg Tyr Phe Ala Phe IleAla Asp Ala Leu Ile Thr Thr Leu Lys Pro 1265 1270 1275 1280 AGA TAT GAAGAT ACT AAT ATA AGA ATA AAT TTA GAT AGT AAT ACT AGA 3888 Arg Tyr Glu AspThr Asn Ile Arg Ile Asn Leu Asp Ser Asn Thr Arg 1285 1290 1295 AGT TTTATA GTT CCA ATA ATA ACT ACA GAA TAT ATA AGA GAA AAA TTA 3936 Ser Phe IleVal Pro Ile Ile Thr Thr Glu Tyr Ile Arg Glu Lys Leu 1300 1305 1310 TCATAT TCT TTC TAT GGT TCA GGA GGA ACT TAT GCA TTG TCT CTT TCT 3984 Ser TyrSer Phe Tyr Gly Ser Gly Gly Thr Tyr Ala Leu Ser Leu Ser 1315 1320 1325CAA TAT AAT ATG GGT ATA AAT ATA GAA TTA AGT GAA AGT GAT GTT TGG 4032 GlnTyr Asn Met Gly Ile Asn Ile Glu Leu Ser Glu Ser Asp Val Trp 1330 13351340 ATT ATA GAT GTT GAT AAT GTT GTG AGA GAT GTA ACT ATA GAA TCT GAT4080 Ile Ile Asp Val Asp Asn Val Val Arg Asp Val Thr Ile Glu Ser Asp1345 1350 1355 1360 AAA ATT AAA AAA GGT GAT TTA ATA GAA GGT ATT TTA TCTACA CTA AGT 4128 Lys Ile Lys Lys Gly Asp Leu Ile Glu Gly Ile Leu Ser ThrLeu Ser 1365 1370 1375 ATT GAA GAG AAT AAA ATT ATC TTA AAT AGC CAT GAGATT AAT TTT TCT 4176 Ile Glu Glu Asn Lys Ile Ile Leu Asn Ser His Glu IleAsn Phe Ser 1380 1385 1390 GGT GAG GTA AAT GGA AGT AAT GGA TTT GTT TCTTTA ACA TTT TCA ATT 4224 Gly Glu Val Asn Gly Ser Asn Gly Phe Val Ser LeuThr Phe Ser Ile 1395 1400 1405 TTA GAA GGA ATA AAT GCA ATT ATA GAA GTTGAT TTA TTA TCT AAA TCA 4272 Leu Glu Gly Ile Asn Ala Ile Ile Glu Val AspLeu Leu Ser Lys Ser 1410 1415 1420 TAT AAA TTA CTT ATT TCT GGC GAA TTAAAA ATA TTG ATG TTA AAT TCA 4320 Tyr Lys Leu Leu Ile Ser Gly Glu Leu LysIle Leu Met Leu Asn Ser 1425 1430 1435 1440 AAT CAT ATT CAA CAG AAA ATAGAT TAT ATA GGA TTC AAT AGC GAA TTA 4368 Asn His Ile Gln Gln Lys Ile AspTyr Ile Gly Phe Asn Ser Glu Leu 1445 1450 1455 CAG AAA AAT ATA CCA TATAGC TTT GTA GAT AGT GAA GGA AAA GAG AAT 4416 Gln Lys Asn Ile Pro Tyr SerPhe Val Asp Ser Glu Gly Lys Glu Asn 1460 1465 1470 GGT TTT ATT AAT GGTTCA ACA AAA GAA GGT TTA TTT GTA TCT GAA TTA 4464 Gly Phe Ile Asn Gly SerThr Lys Glu Gly Leu Phe Val Ser Glu Leu 1475 1480 1485 CCT GAT GTA GTTCTT ATA AGT AAG GTT TAT ATG GAT GAT AGT AAG CCT 4512 Pro Asp Val Val LeuIle Ser Lys Val Tyr Met Asp Asp Ser Lys Pro 1490 1495 1500 TCA TTT GGATAT TAT AGT AAT AAT TTG AAA GAT GTC AAA GTT ATA ACT 4560 Ser Phe Gly TyrTyr Ser Asn Asn Leu Lys Asp Val Lys Val Ile Thr 1505 1510 1515 1520 AAAGAT AAT GTT AAT ATA TTA ACA GGT TAT TAT CTT AAG GAT GAT ATA 4608 Lys AspAsn Val Asn Ile Leu Thr Gly Tyr Tyr Leu Lys Asp Asp Ile 1525 1530 1535AAA ATC TCT CTT TCT TTG ACT CTA CAA GAT GAA AAA ACT ATA AAG TTA 4656 LysIle Ser Leu Ser Leu Thr Leu Gln Asp Glu Lys Thr Ile Lys Leu 1540 15451550 AAT AGT GTG CAT TTA GAT GAA AGT GGA GTA GCT GAG ATT TTG AAG TTC4704 Asn Ser Val His Leu Asp Glu Ser Gly Val Ala Glu Ile Leu Lys Phe1555 1560 1565 ATG AAT AGA AAA GGT AAT ACA AAT ACT TCA GAT TCT TTA ATGAGC TTT 4752 Met Asn Arg Lys Gly Asn Thr Asn Thr Ser Asp Ser Leu Met SerPhe 1570 1575 1580 TTA GAA AGT ATG AAT ATA AAA AGT ATT TTC GTT AAT TTCTTA CAA TCT 4800 Leu Glu Ser Met Asn Ile Lys Ser Ile Phe Val Asn Phe LeuGln Ser 1585 1590 1595 1600 AAT ATT AAG TTT ATA TTA GAT GCT AAT TTT ATAATA AGT GGT ACT ACT 4848 Asn Ile Lys Phe Ile Leu Asp Ala Asn Phe Ile IleSer Gly Thr Thr 1605 1610 1615 TCT ATT GGC CAA TTT GAG TTT ATT TGT GATGAA AAT GAT AAT ATA CAA 4896 Ser Ile Gly Gln Phe Glu Phe Ile Cys Asp GluAsn Asp Asn Ile Gln 1620 1625 1630 CCA TAT TTC ATT AAG TTT AAT ACA CTAGAA ACT AAT TAT ACT TTA TAT 4944 Pro Tyr Phe Ile Lys Phe Asn Thr Leu GluThr Asn Tyr Thr Leu Tyr 1635 1640 1645 GTA GGA AAT AGA CAA AAT ATG ATAGTG GAA CCA AAT TAT GAT TTA GAT 4992 Val Gly Asn Arg Gln Asn Met Ile ValGlu Pro Asn Tyr Asp Leu Asp 1650 1655 1660 GAT TCT GGA GAT ATA TCT TCAACT GTT ATC AAT TTC TCT CAA AAG TAT 5040 Asp Ser Gly Asp Ile Ser Ser ThrVal Ile Asn Phe Ser Gln Lys Tyr 1665 1670 1675 1680 CTT TAT GGA ATA GACAGT TGT GTT AAT AAA GTT GTA ATT TCA CCA AAT 5088 Leu Tyr Gly Ile Asp SerCys Val Asn Lys Val Val Ile Ser Pro Asn 1685 1690 1695 ATT TAT ACA GATGAA ATA AAT ATA ACG CCT GTA TAT GAA ACA AAT AAT 5136 Ile Tyr Thr Asp GluIle Asn Ile Thr Pro Val Tyr Glu Thr Asn Asn 1700 1705 1710 ACT TAT CCAGAA GTT ATT GTA TTA GAT GCA AAT TAT ATA AAT GAA AAA 5184 Thr Tyr Pro GluVal Ile Val Leu Asp Ala Asn Tyr Ile Asn Glu Lys 1715 1720 1725 ATA AATGTT AAT ATC AAT GAT CTA TCT ATA CGA TAT GTA TGG AGT AAT 5232 Ile Asn ValAsn Ile Asn Asp Leu Ser Ile Arg Tyr Val Trp Ser Asn 1730 1735 1740 GATGGT AAT GAT TTT ATT CTT ATG TCA ACT AGT GAA GAA AAT AAG GTG 5280 Asp GlyAsn Asp Phe Ile Leu Met Ser Thr Ser Glu Glu Asn Lys Val 1745 1750 17551760 TCA CAA GTT AAA ATA AGA TTC GTT AAT GTT TTT AAA GAT AAG ACT TTG5328 Ser Gln Val Lys Ile Arg Phe Val Asn Val Phe Lys Asp Lys Thr Leu1765 1770 1775 GCA AAT AAG CTA TCT TTT AAC TTT AGT GAT AAA CAA GAT GTACCT GTA 5376 Ala Asn Lys Leu Ser Phe Asn Phe Ser Asp Lys Gln Asp Val ProVal 1780 1785 1790 AGT GAA ATA ATC TTA TCA TTT ACA CCT TCA TAT TAT GAGGAT GGA TTG 5424 Ser Glu Ile Ile Leu Ser Phe Thr Pro Ser Tyr Tyr Glu AspGly Leu 1795 1800 1805 ATT GGC TAT GAT TTG GGT CTA GTT TCT TTA TAT AATGAG AAA TTT TAT 5472 Ile Gly Tyr Asp Leu Gly Leu Val Ser Leu Tyr Asn GluLys Phe Tyr 1810 1815 1820 ATT AAT AAC TTT GGA ATG ATG GTA TCT GGA TTAATA TAT ATT AAT GAT 5520 Ile Asn Asn Phe Gly Met Met Val Ser Gly Leu IleTyr Ile Asn Asp 1825 1830 1835 1840 TCA TTA TAT TAT TTT AAA CCA CCA GTAAAT AAT TTG ATA ACT GGA TTT 5568 Ser Leu Tyr Tyr Phe Lys Pro Pro Val AsnAsn Leu Ile Thr Gly Phe 1845 1850 1855 GTG ACT GTA GGC GAT GAT AAA TACTAC TTT AAT CCA ATT AAT GGT GGA 5616 Val Thr Val Gly Asp Asp Lys Tyr TyrPhe Asn Pro Ile Asn Gly Gly 1860 1865 1870 GCT GCT TCA ATT GGA GAG ACAATA ATT GAT GAC AAA AAT TAT TAT TTC 5664 Ala Ala Ser Ile Gly Glu Thr IleIle Asp Asp Lys Asn Tyr Tyr Phe 1875 1880 1885 AAC CAA AGT GGA GTG TTACAA ACA GGT GTA TTT AGT ACA GAA GAT GGA 5712 Asn Gln Ser Gly Val Leu GlnThr Gly Val Phe Ser Thr Glu Asp Gly 1890 1895 1900 TTT AAA TAT TTT GCCCCA GCT AAT ACA CTT GAT GAA AAC CTA GAA GGA 5760 Phe Lys Tyr Phe Ala ProAla Asn Thr Leu Asp Glu Asn Leu Glu Gly 1905 1910 1915 1920 GAA GCA ATTGAT TTT ACT GGA AAA TTA ATT ATT GAC GAA AAT ATT TAT 5808 Glu Ala Ile AspPhe Thr Gly Lys Leu Ile Ile Asp Glu Asn Ile Tyr 1925 1930 1935 TAT TTTGAT GAT AAT TAT AGA GGA GCT GTA GAA TGG AAA GAA TTA GAT 5856 Tyr Phe AspAsp Asn Tyr Arg Gly Ala Val Glu Trp Lys Glu Leu Asp 1940 1945 1950 GGTGAA ATG CAC TAT TTT AGC CCA GAA ACA GGT AAA GCT TTT AAA GGT 5904 Gly GluMet His Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe Lys Gly 1955 1960 1965CTA AAT CAA ATA GGT GAT TAT AAA TAC TAT TTC AAT TCT GAT GGA GTT 5952 LeuAsn Gln Ile Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp Gly Val 1970 19751980 ATG CAA AAA GGA TTT GTT AGT ATA AAT GAT AAT AAA CAC TAT TTT GAT6000 Met Gln Lys Gly Phe Val Ser Ile Asn Asp Asn Lys His Tyr Phe Asp1985 1990 1995 2000 GAT TCT GGT GTT ATG AAA GTA GGT TAC ACT GAA ATA GATGGC AAG CAT 6048 Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu Ile Asp GlyLys His 2005 2010 2015 TTC TAC TTT GCT GAA AAC GGA GAA ATG CAA ATA GGAGTA TTT AAT ACA 6096 Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly ValPhe Asn Thr 2020 2025 2030 GAA GAT GGA TTT AAA TAT TTT GCT CAT CAT AATGAA GAT TTA GGA AAT 6144 Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn GluAsp Leu Gly Asn 2035 2040 2045 GAA GAA GGT GAA GAA ATC TCA TAT TCT GGTATA TTA AAT TTC AAT AAT 6192 Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly IleLeu Asn Phe Asn Asn 2050 2055 2060 AAA ATT TAC TAT TTT GAT GAT TCA TTTACA GCT GTA GTT GGA TGG AAA 6240 Lys Ile Tyr Tyr Phe Asp Asp Ser Phe ThrAla Val Val Gly Trp Lys 2065 2070 2075 2080 GAT TTA GAG GAT GGT TCA AAGTAT TAT TTT GAT GAA GAT ACA GCA GAA 6288 Asp Leu Glu Asp Gly Ser Lys TyrTyr Phe Asp Glu Asp Thr Ala Glu 2085 2090 2095 GCA TAT ATA GGT TTG TCATTA ATA AAT GAT GGT CAA TAT TAT TTT AAT 6336 Ala Tyr Ile Gly Leu Ser LeuIle Asn Asp Gly Gln Tyr Tyr Phe Asn 2100 2105 2110 GAT GAT GGA ATT ATGCAA GTT GGA TTT GTC ACT ATA AAT GAT AAA GTC 6384 Asp Asp Gly Ile Met GlnVal Gly Phe Val Thr Ile Asn Asp Lys Val 2115 2120 2125 TTC TAC TTC TCTGAC TCT GGA ATT ATA GAA TCT GGA GTA CAA AAC ATA 6432 Phe Tyr Phe Ser AspSer Gly Ile Ile Glu Ser Gly Val Gln Asn Ile 2130 2135 2140 GAT GAC AATTAT TTC TAT ATA GAT GAT AAT GGT ATA GTT CAA ATT GGT 6480 Asp Asp Asn TyrPhe Tyr Ile Asp Asp Asn Gly Ile Val Gln Ile Gly 2145 2150 2155 2160 GTATTT GAT ACT TCA GAT GGA TAT AAA TAT TTT GCA CCT GCT AAT ACT 6528 Val PheAsp Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr 2165 2170 2175GTA AAT GAT AAT ATT TAC GGA CAA GCA GTT GAA TAT AGT GGT TTA GTT 6576 ValAsn Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val 2180 21852190 AGA GTT GGG GAA GAT GTA TAT TAT TTT GGA GAA ACA TAT ACA ATT GAG6624 Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile Glu2195 2200 2205 ACT GGA TGG ATA TAT GAT ATG GAA AAT GAA AGT GAT AAA TATTAT TTC 6672 Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr TyrPhe 2210 2215 2220 AAT CCA GAA ACT AAA AAA GCA TGC AAA GGT ATT AAT TTAATT GAT GAT 6720 Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu IleAsp Asp 2225 2230 2235 2240 ATA AAA TAT TAT TTT GAT GAG AAG GGC ATA ATGAGA ACG GGT CTT ATA 6768 Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met ArgThr Gly Leu Ile 2245 2250 2255 TCA TTT GAA AAT AAT AAT TAT TAC TTT AATGAG AAT GGT GAA ATG CAA 6816 Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn GluAsn Gly Glu Met Gln 2260 2265 2270 TTT GGT TAT ATA AAT ATA GAA GAT AAGATG TTC TAT TTT GGT GAA GAT 6864 Phe Gly Tyr Ile Asn Ile Glu Asp Lys MetPhe Tyr Phe Gly Glu Asp 2275 2280 2285 GGT GTC ATG CAG ATT GGA GTA TTTAAT ACA CCA GAT GGA TTT AAA TAC 6912 Gly Val Met Gln Ile Gly Val Phe AsnThr Pro Asp Gly Phe Lys Tyr 2290 2295 2300 TTT GCA CAT CAA AAT ACT TTGGAT GAG AAT TTT GAG GGA GAA TCA ATA 6960 Phe Ala His Gln Asn Thr Leu AspGlu Asn Phe Glu Gly Glu Ser Ile 2305 2310 2315 2320 AAC TAT ACT GGT TGGTTA GAT TTA GAT GAA AAG AGA TAT TAT TTT ACA 7008 Asn Tyr Thr Gly Trp LeuAsp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr 2325 2330 2335 GAT GAA TAT ATTGCA GCA ACT GGT TCA GTT ATT ATT GAT GGT GAG GAG 7056 Asp Glu Tyr Ile AlaAla Thr Gly Ser Val Ile Ile Asp Gly Glu Glu 2340 2345 2350 TAT TAT TTTGAT CCT GAT ACA GCT CAA TTA GTG ATT AGT GAA 7098 Tyr Tyr Phe Asp Pro AspThr Ala Gln Leu Val Ile Ser Glu 2355 2360 2365 TAG 7101 2366 amino acidsamino acid linear protein 10 Met Ser Leu Val Asn Arg Lys Gln Leu Glu LysMet Ala Asn Val Arg 1 5 10 15 Phe Arg Thr Gln Glu Asp Glu Tyr Val AlaIle Leu Asp Ala Leu Glu 20 25 30 Glu Tyr His Asn Met Ser Glu Asn Thr ValVal Glu Lys Tyr Leu Lys 35 40 45 Leu Lys Asp Ile Asn Ser Leu Thr Asp IleTyr Ile Asp Thr Tyr Lys 50 55 60 Lys Ser Gly Arg Asn Lys Ala Leu Lys LysPhe Lys Glu Tyr Leu Val 65 70 75 80 Thr Glu Val Leu Glu Leu Lys Asn AsnAsn Leu Thr Pro Val Glu Lys 85 90 95 Asn Leu His Phe Val Trp Ile Gly GlyGln Ile Asn Asp Thr Ala Ile 100 105 110 Asn Tyr Ile Asn Gln Trp Lys AspVal Asn Ser Asp Tyr Asn Val Asn 115 120 125 Val Phe Tyr Asp Ser Asn AlaPhe Leu Ile Asn Thr Leu Lys Lys Thr 130 135 140 Val Val Glu Ser Ala IleAsn Asp Thr Leu Glu Ser Phe Arg Glu Asn 145 150 155 160 Leu Asn Asp ProArg Phe Asp Tyr Asn Lys Phe Phe Arg Lys Arg Met 165 170 175 Glu Ile IleTyr Asp Lys Gln Lys Asn Phe Ile Asn Tyr Tyr Lys Ala 180 185 190 Gln ArgGlu Glu Asn Pro Glu Leu Ile Ile Asp Asp Ile Val Lys Thr 195 200 205 TyrLeu Ser Asn Glu Tyr Ser Lys Glu Ile Asp Glu Leu Asn Thr Tyr 210 215 220Ile Glu Glu Ser Leu Asn Lys Ile Thr Gln Asn Ser Gly Asn Asp Val 225 230235 240 Arg Asn Phe Glu Glu Phe Lys Asn Gly Glu Ser Phe Asn Leu Tyr Glu245 250 255 Gln Glu Leu Val Glu Arg Trp Asn Leu Ala Ala Ala Ser Asp IleLeu 260 265 270 Arg Ile Ser Ala Leu Lys Glu Ile Gly Gly Met Tyr Leu AspVal Asp 275 280 285 Met Leu Pro Gly Ile Gln Pro Asp Leu Phe Glu Ser IleGlu Lys Pro 290 295 300 Ser Ser Val Thr Val Asp Phe Trp Glu Met Thr LysLeu Glu Ala Ile 305 310 315 320 Met Lys Tyr Lys Glu Tyr Ile Pro Glu TyrThr Ser Glu His Phe Asp 325 330 335 Met Leu Asp Glu Glu Val Gln Ser SerPhe Glu Ser Val Leu Ala Ser 340 345 350 Lys Ser Asp Lys Ser Glu Ile PheSer Ser Leu Gly Asp Met Glu Ala 355 360 365 Ser Pro Leu Glu Val Lys IleAla Phe Asn Ser Lys Gly Ile Ile Asn 370 375 380 Gln Gly Leu Ile Ser ValLys Asp Ser Tyr Cys Ser Asn Leu Ile Val 385 390 395 400 Lys Gln Ile GluAsn Arg Tyr Lys Ile Leu Asn Asn Ser Leu Asn Pro 405 410 415 Ala Ile SerGlu Asp Asn Asp Phe Asn Thr Thr Thr Asn Thr Phe Ile 420 425 430 Asp SerIle Met Ala Glu Ala Asn Ala Asp Asn Gly Arg Phe Met Met 435 440 445 GluLeu Gly Lys Tyr Leu Arg Val Gly Phe Phe Pro Asp Val Lys Thr 450 455 460Thr Ile Asn Leu Ser Gly Pro Glu Ala Tyr Ala Ala Ala Tyr Gln Asp 465 470475 480 Leu Leu Met Phe Lys Glu Gly Ser Met Asn Ile His Leu Ile Glu Ala485 490 495 Asp Leu Arg Asn Phe Glu Ile Ser Lys Thr Asn Ile Ser Gln SerThr 500 505 510 Glu Gln Glu Met Ala Ser Leu Trp Ser Phe Asp Asp Ala ArgAla Lys 515 520 525 Ala Gln Phe Glu Glu Tyr Lys Arg Asn Tyr Phe Glu GlySer Leu Gly 530 535 540 Glu Asp Asp Asn Leu Asp Phe Ser Gln Asn Ile ValVal Asp Lys Glu 545 550 555 560 Tyr Leu Leu Glu Lys Ile Ser Ser Leu AlaArg Ser Ser Glu Arg Gly 565 570 575 Tyr Ile His Tyr Ile Val Gln Leu GlnGly Asp Lys Ile Ser Tyr Glu 580 585 590 Ala Ala Cys Asn Leu Phe Ala LysThr Pro Tyr Asp Ser Val Leu Phe 595 600 605 Gln Lys Asn Ile Glu Asp SerGlu Ile Ala Tyr Tyr Tyr Asn Pro Gly 610 615 620 Asp Gly Glu Ile Gln GluIle Asp Lys Tyr Lys Ile Pro Ser Ile Ile 625 630 635 640 Ser Asp Arg ProLys Ile Lys Leu Thr Phe Ile Gly His Gly Lys Asp 645 650 655 Glu Phe AsnThr Asp Ile Phe Ala Gly Phe Asp Val Asp Ser Leu Ser 660 665 670 Thr GluIle Glu Ala Ala Ile Asp Leu Ala Lys Glu Asp Ile Ser Pro 675 680 685 LysSer Ile Glu Ile Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr Ser 690 695 700Ile Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Lys Val Lys 705 710715 720 Asp Lys Ile Ser Glu Leu Met Pro Ser Ile Ser Gln Asp Ser Ile Ile725 730 735 Val Ser Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly ArgArg 740 745 750 Glu Leu Leu Asp His Ser Gly Glu Trp Ile Asn Lys Glu GluSer Ile 755 760 765 Ile Lys Asp Ile Ser Ser Lys Glu Tyr Ile Ser Phe AsnPro Lys Glu 770 775 780 Asn Lys Ile Thr Val Lys Ser Lys Asn Leu Pro GluLeu Ser Thr Leu 785 790 795 800 Leu Gln Glu Ile Arg Asn Asn Ser Asn SerSer Asp Ile Glu Leu Glu 805 810 815 Glu Lys Val Met Leu Thr Glu Cys GluIle Asn Val Ile Ser Asn Ile 820 825 830 Asp Thr Gln Ile Val Glu Glu ArgIle Glu Glu Ala Lys Asn Leu Thr 835 840 845 Ser Asp Ser Ile Asn Tyr IleLys Asp Glu Phe Lys Leu Ile Glu Ser 850 855 860 Ile Ser Asp Ala Leu CysAsp Leu Lys Gln Gln Asn Glu Leu Glu Asp 865 870 875 880 Ser His Phe IleSer Phe Glu Asp Ile Ser Glu Thr Asp Glu Gly Phe 885 890 895 Ser Ile ArgPhe Ile Asn Lys Glu Thr Gly Glu Ser Ile Phe Val Glu 900 905 910 Thr GluLys Thr Ile Phe Ser Glu Tyr Ala Asn His Ile Thr Glu Glu 915 920 925 IleSer Lys Ile Lys Gly Thr Ile Phe Asp Thr Val Asn Gly Lys Leu 930 935 940Val Lys Lys Val Asn Leu Asp Thr Thr His Glu Val Asn Thr Leu Asn 945 950955 960 Ala Ala Phe Phe Ile Gln Ser Leu Ile Glu Tyr Asn Ser Ser Lys Glu965 970 975 Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gln Val Tyr AlaGln 980 985 990 Leu Phe Ser Thr Gly Leu Asn Thr Ile Thr Asp Ala Ala LysVal Val 995 1000 1005 Glu Leu Val Ser Thr Ala Leu Asp Glu Thr Ile AspLeu Leu Pro Thr 1010 1015 1020 Leu Ser Glu Gly Leu Pro Ile Ile Ala ThrIle Ile Asp Gly Val Ser 1025 1030 1035 1040 Leu Gly Ala Ala Ile Lys GluLeu Ser Glu Thr Ser Asp Pro Leu Leu 1045 1050 1055 Arg Gln Glu Ile GluAla Lys Ile Gly Ile Met Ala Val Asn Leu Thr 1060 1065 1070 Thr Ala ThrThr Ala Ile Ile Thr Ser Ser Leu Gly Ile Ala Ser Gly 1075 1080 1085 PheSer Ile Leu Leu Val Pro Leu Ala Gly Ile Ser Ala Gly Ile Pro 1090 10951100 Ser Leu Val Asn Asn Glu Leu Val Leu Arg Asp Lys Ala Thr Lys Val1105 1110 1115 1120 Val Asp Tyr Phe Lys His Val Ser Leu Val Glu Thr GluGly Val Phe 1125 1130 1135 Thr Leu Leu Asp Asp Lys Ile Met Met Pro GlnAsp Asp Leu Val Ile 1140 1145 1150 Ser Glu Ile Asp Phe Asn Asn Asn SerIle Val Leu Gly Lys Cys Glu 1155 1160 1165 Ile Trp Arg Met Glu Gly GlySer Gly His Thr Val Thr Asp Asp Ile 1170 1175 1180 Asp His Phe Phe SerAla Pro Ser Ile Thr Tyr Arg Glu Pro His Leu 1185 1190 1195 1200 Ser IleTyr Asp Val Leu Glu Val Gln Lys Glu Glu Leu Asp Leu Ser 1205 1210 1215Lys Asp Leu Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Ala Trp 12201225 1230 Glu Thr Gly Trp Thr Pro Gly Leu Arg Ser Leu Glu Asn Asp GlyThr 1235 1240 1245 Lys Leu Leu Asp Arg Ile Arg Asp Asn Tyr Glu Gly GluPhe Tyr Trp 1250 1255 1260 Arg Tyr Phe Ala Phe Ile Ala Asp Ala Leu IleThr Thr Leu Lys Pro 1265 1270 1275 1280 Arg Tyr Glu Asp Thr Asn Ile ArgIle Asn Leu Asp Ser Asn Thr Arg 1285 1290 1295 Ser Phe Ile Val Pro IleIle Thr Thr Glu Tyr Ile Arg Glu Lys Leu 1300 1305 1310 Ser Tyr Ser PheTyr Gly Ser Gly Gly Thr Tyr Ala Leu Ser Leu Ser 1315 1320 1325 Gln TyrAsn Met Gly Ile Asn Ile Glu Leu Ser Glu Ser Asp Val Trp 1330 1335 1340Ile Ile Asp Val Asp Asn Val Val Arg Asp Val Thr Ile Glu Ser Asp 13451350 1355 1360 Lys Ile Lys Lys Gly Asp Leu Ile Glu Gly Ile Leu Ser ThrLeu Ser 1365 1370 1375 Ile Glu Glu Asn Lys Ile Ile Leu Asn Ser His GluIle Asn Phe Ser 1380 1385 1390 Gly Glu Val Asn Gly Ser Asn Gly Phe ValSer Leu Thr Phe Ser Ile 1395 1400 1405 Leu Glu Gly Ile Asn Ala Ile IleGlu Val Asp Leu Leu Ser Lys Ser 1410 1415 1420 Tyr Lys Leu Leu Ile SerGly Glu Leu Lys Ile Leu Met Leu Asn Ser 1425 1430 1435 1440 Asn His IleGln Gln Lys Ile Asp Tyr Ile Gly Phe Asn Ser Glu Leu 1445 1450 1455 GlnLys Asn Ile Pro Tyr Ser Phe Val Asp Ser Glu Gly Lys Glu Asn 1460 14651470 Gly Phe Ile Asn Gly Ser Thr Lys Glu Gly Leu Phe Val Ser Glu Leu1475 1480 1485 Pro Asp Val Val Leu Ile Ser Lys Val Tyr Met Asp Asp SerLys Pro 1490 1495 1500 Ser Phe Gly Tyr Tyr Ser Asn Asn Leu Lys Asp ValLys Val Ile Thr 1505 1510 1515 1520 Lys Asp Asn Val Asn Ile Leu Thr GlyTyr Tyr Leu Lys Asp Asp Ile 1525 1530 1535 Lys Ile Ser Leu Ser Leu ThrLeu Gln Asp Glu Lys Thr Ile Lys Leu 1540 1545 1550 Asn Ser Val His LeuAsp Glu Ser Gly Val Ala Glu Ile Leu Lys Phe 1555 1560 1565 Met Asn ArgLys Gly Asn Thr Asn Thr Ser Asp Ser Leu Met Ser Phe 1570 1575 1580 LeuGlu Ser Met Asn Ile Lys Ser Ile Phe Val Asn Phe Leu Gln Ser 1585 15901595 1600 Asn Ile Lys Phe Ile Leu Asp Ala Asn Phe Ile Ile Ser Gly ThrThr 1605 1610 1615 Ser Ile Gly Gln Phe Glu Phe Ile Cys Asp Glu Asn AspAsn Ile Gln 1620 1625 1630 Pro Tyr Phe Ile Lys Phe Asn Thr Leu Glu ThrAsn Tyr Thr Leu Tyr 1635 1640 1645 Val Gly Asn Arg Gln Asn Met Ile ValGlu Pro Asn Tyr Asp Leu Asp 1650 1655 1660 Asp Ser Gly Asp Ile Ser SerThr Val Ile Asn Phe Ser Gln Lys Tyr 1665 1670 1675 1680 Leu Tyr Gly IleAsp Ser Cys Val Asn Lys Val Val Ile Ser Pro Asn 1685 1690 1695 Ile TyrThr Asp Glu Ile Asn Ile Thr Pro Val Tyr Glu Thr Asn Asn 1700 1705 1710Thr Tyr Pro Glu Val Ile Val Leu Asp Ala Asn Tyr Ile Asn Glu Lys 17151720 1725 Ile Asn Val Asn Ile Asn Asp Leu Ser Ile Arg Tyr Val Trp SerAsn 1730 1735 1740 Asp Gly Asn Asp Phe Ile Leu Met Ser Thr Ser Glu GluAsn Lys Val 1745 1750 1755 1760 Ser Gln Val Lys Ile Arg Phe Val Asn ValPhe Lys Asp Lys Thr Leu 1765 1770 1775 Ala Asn Lys Leu Ser Phe Asn PheSer Asp Lys Gln Asp Val Pro Val 1780 1785 1790 Ser Glu Ile Ile Leu SerPhe Thr Pro Ser Tyr Tyr Glu Asp Gly Leu 1795 1800 1805 Ile Gly Tyr AspLeu Gly Leu Val Ser Leu Tyr Asn Glu Lys Phe Tyr 1810 1815 1820 Ile AsnAsn Phe Gly Met Met Val Ser Gly Leu Ile Tyr Ile Asn Asp 1825 1830 18351840 Ser Leu Tyr Tyr Phe Lys Pro Pro Val Asn Asn Leu Ile Thr Gly Phe1845 1850 1855 Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn Pro Ile AsnGly Gly 1860 1865 1870 Ala Ala Ser Ile Gly Glu Thr Ile Ile Asp Asp LysAsn Tyr Tyr Phe 1875 1880 1885 Asn Gln Ser Gly Val Leu Gln Thr Gly ValPhe Ser Thr Glu Asp Gly 1890 1895 1900 Phe Lys Tyr Phe Ala Pro Ala AsnThr Leu Asp Glu Asn Leu Glu Gly 1905 1910 1915 1920 Glu Ala Ile Asp PheThr Gly Lys Leu Ile Ile Asp Glu Asn Ile Tyr 1925 1930 1935 Tyr Phe AspAsp Asn Tyr Arg Gly Ala Val Glu Trp Lys Glu Leu Asp 1940 1945 1950 GlyGlu Met His Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe Lys Gly 1955 19601965 Leu Asn Gln Ile Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp Gly Val1970 1975 1980 Met Gln Lys Gly Phe Val Ser Ile Asn Asp Asn Lys His TyrPhe Asp 1985 1990 1995 2000 Asp Ser Gly Val Met Lys Val Gly Tyr Thr GluIle Asp Gly Lys His 2005 2010 2015 Phe Tyr Phe Ala Glu Asn Gly Glu MetGln Ile Gly Val Phe Asn Thr 2020 2025 2030 Glu Asp Gly Phe Lys Tyr PheAla His His Asn Glu Asp Leu Gly Asn 2035 2040 2045 Glu Glu Gly Glu GluIle Ser Tyr Ser Gly Ile Leu Asn Phe Asn Asn 2050 2055 2060 Lys Ile TyrTyr Phe Asp Asp Ser Phe Thr Ala Val Val Gly Trp Lys 2065 2070 2075 2080Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Ala Glu 20852090 2095 Ala Tyr Ile Gly Leu Ser Leu Ile Asn Asp Gly Gln Tyr Tyr PheAsn 2100 2105 2110 Asp Asp Gly Ile Met Gln Val Gly Phe Val Thr Ile AsnAsp Lys Val 2115 2120 2125 Phe Tyr Phe Ser Asp Ser Gly Ile Ile Glu SerGly Val Gln Asn Ile 2130 2135 2140 Asp Asp Asn Tyr Phe Tyr Ile Asp AspAsn Gly Ile Val Gln Ile Gly 2145 2150 2155 2160 Val Phe Asp Thr Ser AspGly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr 2165 2170 2175 Val Asn Asp AsnIle Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val 2180 2185 2190 Arg ValGly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile Glu 2195 2200 2205Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe 22102215 2220 Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu Ile AspAsp 2225 2230 2235 2240 Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met ArgThr Gly Leu Ile 2245 2250 2255 Ser Phe Glu Asn Asn Asn Tyr Tyr Phe AsnGlu Asn Gly Glu Met Gln 2260 2265 2270 Phe Gly Tyr Ile Asn Ile Glu AspLys Met Phe Tyr Phe Gly Glu Asp 2275 2280 2285 Gly Val Met Gln Ile GlyVal Phe Asn Thr Pro Asp Gly Phe Lys Tyr 2290 2295 2300 Phe Ala His GlnAsn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser Ile 2305 2310 2315 2320 AsnTyr Thr Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr 2325 23302335 Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val Ile Ile Asp Gly Glu Glu2340 2345 2350 Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu Val Ile Ser Glu2355 2360 2365 19 base pairs nucleic acid single linear DNA (genomic) 11TAGAAAAAAT GGCAAATGT 19 21 base pairs nucleic acid single linear DNA(genomic) 12 TTTCATCTTG TAGAGTCAAA G 21 22 base pairs nucleic acidsingle linear DNA (genomic) 13 GATGCCACAA GATGATTTAG TG 22 22 base pairsnucleic acid single linear DNA (genomic) 14 CTAATTGAGC TGTATCAGGA TC 2227 base pairs nucleic acid single linear DNA (genomic) 15 CGGAATTCCTAGAAAAAATG GCAAATG 27 26 base pairs nucleic acid single linear DNA(genomic) 16 GCTCTAGAAT GACCATAAGC TAGCCA 26 27 base pairs nucleic acidsingle linear DNA (genomic) 17 CGGAATTCGA GTTGGTAGAA AGGTGGA 27 27 basepairs nucleic acid single linear DNA (genomic) 18 CGGAATTCGG TTATTATCTTAAGGATG 27 28 base pairs nucleic acid single linear DNA (genomic) 19CGGAATTCTT GATAACTGGA TTTGTGAC 28 511 amino acids amino acid unknownunknown protein 20 Leu Ile Thr Gly Phe Val Thr Val Gly Asp Asp Lys TyrTyr Phe Asn 1 5 10 15 Pro Ile Asn Gly Gly Ala Ala Ser Ile Gly Glu ThrIle Ile Asp Asp 20 25 30 Lys Asn Tyr Tyr Phe Asn Gln Ser Gly Val Leu GlnThr Gly Val Phe 35 40 45 Ser Thr Glu Asp Gly Phe Lys Tyr Phe Ala Pro AlaAsn Thr Leu Asp 50 55 60 Glu Asn Leu Glu Gly Glu Ala Ile Asp Phe Thr GlyLys Leu Ile Ile 65 70 75 80 Asp Glu Asn Ile Tyr Tyr Phe Asp Asp Asn TyrArg Gly Ala Val Glu 85 90 95 Trp Lys Glu Leu Asp Gly Glu Met His Tyr PheSer Pro Glu Thr Gly 100 105 110 Lys Ala Phe Lys Gly Leu Asn Gln Ile GlyAsp Tyr Lys Tyr Tyr Phe 115 120 125 Asn Ser Asp Gly Val Met Gln Lys GlyPhe Val Ser Ile Asn Asp Asn 130 135 140 Lys His Tyr Phe Asp Asp Ser GlyVal Met Lys Val Gly Tyr Thr Glu 145 150 155 160 Ile Asp Gly Lys His PheTyr Phe Ala Glu Asn Gly Glu Met Gln Ile 165 170 175 Gly Val Phe Asn ThrGlu Asp Gly Phe Lys Tyr Phe Ala His His Asn 180 185 190 Glu Asp Leu GlyAsn Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly Ile 195 200 205 Leu Asn PheAsn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser Phe Thr Ala 210 215 220 Val ValGly Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp 225 230 235 240Glu Asp Thr Ala Glu Ala Tyr Ile Gly Leu Ser Leu Ile Asn Asp Gly 245 250255 Gln Tyr Tyr Phe Asn Asp Asp Gly Ile Met Gln Val Gly Phe Val Thr 260265 270 Ile Asn Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly Ile Ile Glu Ser275 280 285 Gly Val Gln Asn Ile Asp Asp Asn Tyr Phe Tyr Ile Asp Asp AsnGly 290 295 300 Ile Val Gln Ile Gly Val Phe Asp Thr Ser Asp Gly Tyr LysTyr Phe 305 310 315 320 Ala Pro Ala Asn Thr Val Asn Asp Asn Ile Tyr GlyGln Ala Val Glu 325 330 335 Tyr Ser Gly Leu Val Arg Val Gly Glu Asp ValTyr Tyr Phe Gly Glu 340 345 350 Thr Tyr Thr Ile Glu Thr Gly Trp Ile TyrAsp Met Glu Asn Glu Ser 355 360 365 Asp Lys Tyr Tyr Phe Asn Pro Glu ThrLys Lys Ala Cys Lys Gly Ile 370 375 380 Asn Leu Ile Asp Asp Ile Lys TyrTyr Phe Asp Glu Lys Gly Ile Met 385 390 395 400 Arg Thr Gly Leu Ile SerPhe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu 405 410 415 Asn Gly Glu Met GlnPhe Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe 420 425 430 Tyr Phe Gly GluAsp Gly Val Met Gln Ile Gly Val Phe Asn Thr Pro 435 440 445 Asp Gly PheLys Tyr Phe Ala His Gln Asn Thr Leu Asp Glu Asn Phe 450 455 460 Glu GlyGlu Ser Ile Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys 465 470 475 480Arg Tyr Tyr Phe Thr Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val Ile 485 490495 Ile Asp Gly Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu 500 505510 608 amino acids amino acid unknown unknown protein 21 Ser Glu GluAsn Lys Val Ser Gln Val Lys Ile Arg Phe Val Asn Val 1 5 10 15 Phe LysAsp Lys Thr Leu Ala Asn Lys Leu Ser Phe Asn Phe Ser Asp 20 25 30 Lys GlnAsp Val Pro Val Ser Glu Ile Ile Leu Ser Phe Thr Pro Ser 35 40 45 Tyr TyrGlu Asp Gly Leu Ile Gly Tyr Asp Leu Gly Leu Val Ser Leu 50 55 60 Tyr AsnGlu Lys Phe Tyr Ile Asn Asn Phe Gly Met Met Val Ser Gly 65 70 75 80 LeuIle Tyr Ile Asn Asp Ser Leu Tyr Tyr Phe Lys Pro Pro Val Asn 85 90 95 AsnLeu Ile Thr Gly Phe Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe 100 105 110Asn Pro Ile Asn Gly Gly Ala Ala Ser Ile Gly Glu Thr Ile Ile Asp 115 120125 Asp Lys Asn Tyr Tyr Phe Asn Gln Ser Gly Val Leu Gln Thr Gly Val 130135 140 Phe Ser Thr Glu Asp Gly Phe Lys Tyr Phe Ala Pro Ala Asn Thr Leu145 150 155 160 Asp Glu Asn Leu Glu Gly Glu Ala Ile Asp Phe Thr Gly LysLeu Ile 165 170 175 Ile Asp Glu Asn Ile Tyr Tyr Phe Asp Asp Asn Tyr ArgGly Ala Val 180 185 190 Glu Trp Lys Glu Leu Asp Gly Glu Met His Tyr PheSer Pro Glu Thr 195 200 205 Gly Lys Ala Phe Lys Gly Leu Asn Gln Ile GlyAsp Tyr Lys Tyr Tyr 210 215 220 Phe Asn Ser Asp Gly Val Met Gln Lys GlyPhe Val Ser Ile Asn Asp 225 230 235 240 Asn Lys His Tyr Phe Asp Asp SerGly Val Met Lys Val Gly Tyr Thr 245 250 255 Glu Ile Asp Gly Lys His PheTyr Phe Ala Glu Asn Gly Glu Met Gln 260 265 270 Ile Gly Val Phe Asn ThrGlu Asp Gly Phe Lys Tyr Phe Ala His His 275 280 285 Asn Glu Asp Leu GlyAsn Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly 290 295 300 Ile Leu Asn PheAsn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser Phe Thr 305 310 315 320 Ala ValVal Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe 325 330 335 AspGlu Asp Thr Ala Glu Ala Tyr Ile Gly Leu Ser Leu Ile Asn Asp 340 345 350Gly Gln Tyr Tyr Phe Asn Asp Asp Gly Ile Met Gln Val Gly Phe Val 355 360365 Thr Ile Asn Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly Ile Ile Glu 370375 380 Ser Gly Val Gln Asn Ile Asp Asp Asn Tyr Phe Tyr Ile Asp Asp Asn385 390 395 400 Gly Ile Val Gln Ile Gly Val Phe Asp Thr Ser Asp Gly TyrLys Tyr 405 410 415 Phe Ala Pro Ala Asn Thr Val Asn Asp Asn Ile Tyr GlyGln Ala Val 420 425 430 Glu Tyr Ser Gly Leu Val Arg Val Gly Glu Asp ValTyr Tyr Phe Gly 435 440 445 Glu Thr Tyr Thr Ile Glu Thr Gly Trp Ile TyrAsp Met Glu Asn Glu 450 455 460 Ser Asp Lys Tyr Tyr Phe Asn Pro Glu ThrLys Lys Ala Cys Lys Gly 465 470 475 480 Ile Asn Leu Ile Asp Asp Ile LysTyr Tyr Phe Asp Glu Lys Gly Ile 485 490 495 Met Arg Thr Gly Leu Ile SerPhe Glu Asn Asn Asn Tyr Tyr Phe Asn 500 505 510 Glu Asn Gly Glu Met GlnPhe Gly Tyr Ile Asn Ile Glu Asp Lys Met 515 520 525 Phe Tyr Phe Gly GluAsp Gly Val Met Gln Ile Gly Val Phe Asn Thr 530 535 540 Pro Asp Gly PheLys Tyr Phe Ala His Gln Asn Thr Leu Asp Glu Asn 545 550 555 560 Phe GluGly Glu Ser Ile Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu 565 570 575 LysArg Tyr Tyr Phe Thr Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val 580 585 590Ile Ile Asp Gly Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu 595 600605 1330 base pairs nucleic acid double linear DNA (genomic) CDS 1..131422 ATG GCT CGT CTG CTG TCT ACC TTC ACT GAA TAC ATC AAG AAC ATC ATC 48Met Ala Arg Leu Leu Ser Thr Phe Thr Glu Tyr Ile Lys Asn Ile Ile 1 5 1015 AAT ACC TCC ATC CTG AAC CTG CGC TAC GAA TCC AAT CAC CTG ATC GAC 96Asn Thr Ser Ile Leu Asn Leu Arg Tyr Glu Ser Asn His Leu Ile Asp 20 25 30CTG TCT CGC TAC GCT TCC AAA ATC AAC ATC GGT TCT AAA GTT AAC TTC 144 LeuSer Arg Tyr Ala Ser Lys Ile Asn Ile Gly Ser Lys Val Asn Phe 35 40 45 GATCCG ATC GAC AAG AAT CAG ATC CAG CTG TTC AAT CTG GAA TCT TCC 192 Asp ProIle Asp Lys Asn Gln Ile Gln Leu Phe Asn Leu Glu Ser Ser 50 55 60 AAA ATCGAA GTT ATC CTG AAG AAT GCT ATC GTA TAC AAC TCT ATG TAC 240 Lys Ile GluVal Ile Leu Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr 65 70 75 80 GAA AACTTC TCC ACC TCC TTC TGG ATC CGT ATC CCG AAA TAC TTC AAC 288 Glu Asn PheSer Thr Ser Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn 85 90 95 TCC ATC TCTCTG AAC AAT GAA TAC ACC ATC ATC AAC TGC ATG GAA AAC 336 Ser Ile Ser LeuAsn Asn Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn 100 105 110 AAT TCT GGTTGG AAA GTA TCT CTG AAC TAC GGT GAA ATC ATC TGG ACT 384 Asn Ser Gly TrpLys Val Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr 115 120 125 CTG CAG GACACT CAG GAA ATC AAA CAG CGT GTT GTA TTC AAA TAC TCT 432 Leu Gln Asp ThrGln Glu Ile Lys Gln Arg Val Val Phe Lys Tyr Ser 130 135 140 CAG ATG ATCAAC ATC TCT GAC TAC ATC AAT CGC TGG ATC TTC GTT ACC 480 Gln Met Ile AsnIle Ser Asp Tyr Ile Asn Arg Trp Ile Phe Val Thr 145 150 155 160 ATC ACCAAC AAT CGT CTG AAT AAC TCC AAA ATC TAC ATC AAC GGC CGT 528 Ile Thr AsnAsn Arg Leu Asn Asn Ser Lys Ile Tyr Ile Asn Gly Arg 165 170 175 CTG ATCGAC CAG AAA CCG ATC TCC AAT CTG GGT AAC ATC CAC GCT TCT 576 Leu Ile AspGln Lys Pro Ile Ser Asn Leu Gly Asn Ile His Ala Ser 180 185 190 AAT AACATC ATG TTC AAA CTG GAC GGT TGT CGT GAC ACT CAC CGC TAC 624 Asn Asn IleMet Phe Lys Leu Asp Gly Cys Arg Asp Thr His Arg Tyr 195 200 205 ATC TGGATC AAA TAC TTC AAT CTG TTC GAC AAA GAA CTG AAC GAA AAA 672 Ile Trp IleLys Tyr Phe Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys 210 215 220 GAA ATCAAA GAC CTG TAC GAC AAC CAG TCC AAT TCT GGT ATC CTG AAA 720 Glu Ile LysAsp Leu Tyr Asp Asn Gln Ser Asn Ser Gly Ile Leu Lys 225 230 235 240 GACTTC TGG GGT GAC TAC CTG CAG TAC GAC AAA CCG TAC TAC ATG CTG 768 Asp PheTrp Gly Asp Tyr Leu Gln Tyr Asp Lys Pro Tyr Tyr Met Leu 245 250 255 AATCTG TAC GAT CCG AAC AAA TAC GTT GAC GTC AAC AAT GTA GGT ATC 816 Asn LeuTyr Asp Pro Asn Lys Tyr Val Asp Val Asn Asn Val Gly Ile 260 265 270 CGCGGT TAC ATG TAC CTG AAA GGT CCG CGT GGT TCT GTT ATG ACT ACC 864 Arg GlyTyr Met Tyr Leu Lys Gly Pro Arg Gly Ser Val Met Thr Thr 275 280 285 AACATC TAC CTG AAC TCT TCC CTG TAC CGT GGT ACC AAA TTC ATC ATC 912 Asn IleTyr Leu Asn Ser Ser Leu Tyr Arg Gly Thr Lys Phe Ile Ile 290 295 300 AAGAAA TAC GCG TCT GGT AAC AAG GAC AAT ATC GTT CGC AAC AAT GAT 960 Lys LysTyr Ala Ser Gly Asn Lys Asp Asn Ile Val Arg Asn Asn Asp 305 310 315 320CGT GTA TAC ATC AAT GTT GTA GTT AAG AAC AAA GAA TAC CGT CTG GCT 1008 ArgVal Tyr Ile Asn Val Val Val Lys Asn Lys Glu Tyr Arg Leu Ala 325 330 335ACC AAT GCT TCT CAG GCT GGT GTA GAA AAG ATC TTG TCT GCT CTG GAA 1056 ThrAsn Ala Ser Gln Ala Gly Val Glu Lys Ile Leu Ser Ala Leu Glu 340 345 350ATC CCG GAC GTT GGT AAT CTG TCT CAG GTA GTT GTA ATG AAA TCC AAG 1104 IlePro Asp Val Gly Asn Leu Ser Gln Val Val Val Met Lys Ser Lys 355 360 365AAC GAC CAG GGT ATC ACT AAC AAA TGC AAA ATG AAT CTG CAG GAC AAC 1152 AsnAsp Gln Gly Ile Thr Asn Lys Cys Lys Met Asn Leu Gln Asp Asn 370 375 380AAT GGT AAC GAT ATC GGT TTC ATC GGT TTC CAC CAG TTC AAC AAT ATC 1200 AsnGly Asn Asp Ile Gly Phe Ile Gly Phe His Gln Phe Asn Asn Ile 385 390 395400 GCT AAA CTG GTT GCT TCC AAC TGG TAC AAT CGT CAG ATC GAA CGT TCC 1248Ala Lys Leu Val Ala Ser Asn Trp Tyr Asn Arg Gln Ile Glu Arg Ser 405 410415 TCT CGC ACT CTG GGT TGC TCT TGG GAG TTC ATC CCG GTT GAT GAC GGT 1296Ser Arg Thr Leu Gly Cys Ser Trp Glu Phe Ile Pro Val Asp Asp Gly 420 425430 TGG GGT GAA CGT CCG CTG TAACCCGGGA AAGCTT 1330 Trp Gly Glu Arg ProLeu 435 438 amino acids amino acid linear protein 23 Met Ala Arg Leu LeuSer Thr Phe Thr Glu Tyr Ile Lys Asn Ile Ile 1 5 10 15 Asn Thr Ser IleLeu Asn Leu Arg Tyr Glu Ser Asn His Leu Ile Asp 20 25 30 Leu Ser Arg TyrAla Ser Lys Ile Asn Ile Gly Ser Lys Val Asn Phe 35 40 45 Asp Pro Ile AspLys Asn Gln Ile Gln Leu Phe Asn Leu Glu Ser Ser 50 55 60 Lys Ile Glu ValIle Leu Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr 65 70 75 80 Glu Asn PheSer Thr Ser Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn 85 90 95 Ser Ile SerLeu Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn 100 105 110 Asn SerGly Trp Lys Val Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr 115 120 125 LeuGln Asp Thr Gln Glu Ile Lys Gln Arg Val Val Phe Lys Tyr Ser 130 135 140Gln Met Ile Asn Ile Ser Asp Tyr Ile Asn Arg Trp Ile Phe Val Thr 145 150155 160 Ile Thr Asn Asn Arg Leu Asn Asn Ser Lys Ile Tyr Ile Asn Gly Arg165 170 175 Leu Ile Asp Gln Lys Pro Ile Ser Asn Leu Gly Asn Ile His AlaSer 180 185 190 Asn Asn Ile Met Phe Lys Leu Asp Gly Cys Arg Asp Thr HisArg Tyr 195 200 205 Ile Trp Ile Lys Tyr Phe Asn Leu Phe Asp Lys Glu LeuAsn Glu Lys 210 215 220 Glu Ile Lys Asp Leu Tyr Asp Asn Gln Ser Asn SerGly Ile Leu Lys 225 230 235 240 Asp Phe Trp Gly Asp Tyr Leu Gln Tyr AspLys Pro Tyr Tyr Met Leu 245 250 255 Asn Leu Tyr Asp Pro Asn Lys Tyr ValAsp Val Asn Asn Val Gly Ile 260 265 270 Arg Gly Tyr Met Tyr Leu Lys GlyPro Arg Gly Ser Val Met Thr Thr 275 280 285 Asn Ile Tyr Leu Asn Ser SerLeu Tyr Arg Gly Thr Lys Phe Ile Ile 290 295 300 Lys Lys Tyr Ala Ser GlyAsn Lys Asp Asn Ile Val Arg Asn Asn Asp 305 310 315 320 Arg Val Tyr IleAsn Val Val Val Lys Asn Lys Glu Tyr Arg Leu Ala 325 330 335 Thr Asn AlaSer Gln Ala Gly Val Glu Lys Ile Leu Ser Ala Leu Glu 340 345 350 Ile ProAsp Val Gly Asn Leu Ser Gln Val Val Val Met Lys Ser Lys 355 360 365 AsnAsp Gln Gly Ile Thr Asn Lys Cys Lys Met Asn Leu Gln Asp Asn 370 375 380Asn Gly Asn Asp Ile Gly Phe Ile Gly Phe His Gln Phe Asn Asn Ile 385 390395 400 Ala Lys Leu Val Ala Ser Asn Trp Tyr Asn Arg Gln Ile Glu Arg Ser405 410 415 Ser Arg Thr Leu Gly Cys Ser Trp Glu Phe Ile Pro Val Asp AspGly 420 425 430 Trp Gly Glu Arg Pro Leu 435 23 amino acids amino acidunknown linear protein 24 Met Gly His His His His His His His His HisHis Ser Ser Gly His 1 5 10 15 Ile Glu Gly Arg His Met Ala 20 1402 basepairs nucleic acid double linear DNA (genomic) CDS 1..1386 25 ATG GGCCAT CAT CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT 48 Met Gly HisHis His His His His His His His His Ser Ser Gly His 1 5 10 15 ATC GAAGGT CGT CAT ATG GCT AGC ATG GCT CGT CTG CTG TCT ACC TTC 96 Ile Glu GlyArg His Met Ala Ser Met Ala Arg Leu Leu Ser Thr Phe 20 25 30 ACT GAA TACATC AAG AAC ATC ATC AAT ACC TCC ATC CTG AAC CTG CGC 144 Thr Glu Tyr IleLys Asn Ile Ile Asn Thr Ser Ile Leu Asn Leu Arg 35 40 45 TAC GAA TCC AATCAC CTG ATC GAC CTG TCT CGC TAC GCT TCC AAA ATC 192 Tyr Glu Ser Asn HisLeu Ile Asp Leu Ser Arg Tyr Ala Ser Lys Ile 50 55 60 AAC ATC GGT TCT AAAGTT AAC TTC GAT CCG ATC GAC AAG AAT CAG ATC 240 Asn Ile Gly Ser Lys ValAsn Phe Asp Pro Ile Asp Lys Asn Gln Ile 65 70 75 80 CAG CTG TTC AAT CTGGAA TCT TCC AAA ATC GAA GTT ATC CTG AAG AAT 288 Gln Leu Phe Asn Leu GluSer Ser Lys Ile Glu Val Ile Leu Lys Asn 85 90 95 GCT ATC GTA TAC AAC TCTATG TAC GAA AAC TTC TCC ACC TCC TTC TGG 336 Ala Ile Val Tyr Asn Ser MetTyr Glu Asn Phe Ser Thr Ser Phe Trp 100 105 110 ATC CGT ATC CCG AAA TACTTC AAC TCC ATC TCT CTG AAC AAT GAA TAC 384 Ile Arg Ile Pro Lys Tyr PheAsn Ser Ile Ser Leu Asn Asn Glu Tyr 115 120 125 ACC ATC ATC AAC TGC ATGGAA AAC AAT TCT GGT TGG AAA GTA TCT CTG 432 Thr Ile Ile Asn Cys Met GluAsn Asn Ser Gly Trp Lys Val Ser Leu 130 135 140 AAC TAC GGT GAA ATC ATCTGG ACT CTG CAG GAC ACT CAG GAA ATC AAA 480 Asn Tyr Gly Glu Ile Ile TrpThr Leu Gln Asp Thr Gln Glu Ile Lys 145 150 155 160 CAG CGT GTT GTA TTCAAA TAC TCT CAG ATG ATC AAC ATC TCT GAC TAC 528 Gln Arg Val Val Phe LysTyr Ser Gln Met Ile Asn Ile Ser Asp Tyr 165 170 175 ATC AAT CGC TGG ATCTTC GTT ACC ATC ACC AAC AAT CGT CTG AAT AAC 576 Ile Asn Arg Trp Ile PheVal Thr Ile Thr Asn Asn Arg Leu Asn Asn 180 185 190 TCC AAA ATC TAC ATCAAC GGC CGT CTG ATC GAC CAG AAA CCG ATC TCC 624 Ser Lys Ile Tyr Ile AsnGly Arg Leu Ile Asp Gln Lys Pro Ile Ser 195 200 205 AAT CTG GGT AAC ATCCAC GCT TCT AAT AAC ATC ATG TTC AAA CTG GAC 672 Asn Leu Gly Asn Ile HisAla Ser Asn Asn Ile Met Phe Lys Leu Asp 210 215 220 GGT TGT CGT GAC ACTCAC CGC TAC ATC TGG ATC AAA TAC TTC AAT CTG 720 Gly Cys Arg Asp Thr HisArg Tyr Ile Trp Ile Lys Tyr Phe Asn Leu 225 230 235 240 TTC GAC AAA GAACTG AAC GAA AAA GAA ATC AAA GAC CTG TAC GAC AAC 768 Phe Asp Lys Glu LeuAsn Glu Lys Glu Ile Lys Asp Leu Tyr Asp Asn 245 250 255 CAG TCC AAT TCTGGT ATC CTG AAA GAC TTC TGG GGT GAC TAC CTG CAG 816 Gln Ser Asn Ser GlyIle Leu Lys Asp Phe Trp Gly Asp Tyr Leu Gln 260 265 270 TAC GAC AAA CCGTAC TAC ATG CTG AAT CTG TAC GAT CCG AAC AAA TAC 864 Tyr Asp Lys Pro TyrTyr Met Leu Asn Leu Tyr Asp Pro Asn Lys Tyr 275 280 285 GTT GAC GTC AACAAT GTA GGT ATC CGC GGT TAC ATG TAC CTG AAA GGT 912 Val Asp Val Asn AsnVal Gly Ile Arg Gly Tyr Met Tyr Leu Lys Gly 290 295 300 CCG CGT GGT TCTGTT ATG ACT ACC AAC ATC TAC CTG AAC TCT TCC CTG 960 Pro Arg Gly Ser ValMet Thr Thr Asn Ile Tyr Leu Asn Ser Ser Leu 305 310 315 320 TAC CGT GGTACC AAA TTC ATC ATC AAG AAA TAC GCG TCT GGT AAC AAG 1008 Tyr Arg Gly ThrLys Phe Ile Ile Lys Lys Tyr Ala Ser Gly Asn Lys 325 330 335 GAC AAT ATCGTT CGC AAC AAT GAT CGT GTA TAC ATC AAT GTT GTA GTT 1056 Asp Asn Ile ValArg Asn Asn Asp Arg Val Tyr Ile Asn Val Val Val 340 345 350 AAG AAC AAAGAA TAC CGT CTG GCT ACC AAT GCT TCT CAG GCT GGT GTA 1104 Lys Asn Lys GluTyr Arg Leu Ala Thr Asn Ala Ser Gln Ala Gly Val 355 360 365 GAA AAG ATCTTG TCT GCT CTG GAA ATC CCG GAC GTT GGT AAT CTG TCT 1152 Glu Lys Ile LeuSer Ala Leu Glu Ile Pro Asp Val Gly Asn Leu Ser 370 375 380 CAG GTA GTTGTA ATG AAA TCC AAG AAC GAC CAG GGT ATC ACT AAC AAA 1200 Gln Val Val ValMet Lys Ser Lys Asn Asp Gln Gly Ile Thr Asn Lys 385 390 395 400 TGC AAAATG AAT CTG CAG GAC AAC AAT GGT AAC GAT ATC GGT TTC ATC 1248 Cys Lys MetAsn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly Phe Ile 405 410 415 GGT TTCCAC CAG TTC AAC AAT ATC GCT AAA CTG GTT GCT TCC AAC TGG 1296 Gly Phe HisGln Phe Asn Asn Ile Ala Lys Leu Val Ala Ser Asn Trp 420 425 430 TAC AATCGT CAG ATC GAA CGT TCC TCT CGC ACT CTG GGT TGC TCT TGG 1344 Tyr Asn ArgGln Ile Glu Arg Ser Ser Arg Thr Leu Gly Cys Ser Trp 435 440 445 GAG TTCATC CCG GTT GAT GAC GGT TGG GGT GAA CGT CCG CTG 1386 Glu Phe Ile Pro ValAsp Asp Gly Trp Gly Glu Arg Pro Leu 450 455 460 TAACCCGGGA AAGCTT 1402462 amino acids amino acid linear protein 26 Met Gly His His His His HisHis His His His His Ser Ser Gly His 1 5 10 15 Ile Glu Gly Arg His MetAla Ser Met Ala Arg Leu Leu Ser Thr Phe 20 25 30 Thr Glu Tyr Ile Lys AsnIle Ile Asn Thr Ser Ile Leu Asn Leu Arg 35 40 45 Tyr Glu Ser Asn His LeuIle Asp Leu Ser Arg Tyr Ala Ser Lys Ile 50 55 60 Asn Ile Gly Ser Lys ValAsn Phe Asp Pro Ile Asp Lys Asn Gln Ile 65 70 75 80 Gln Leu Phe Asn LeuGlu Ser Ser Lys Ile Glu Val Ile Leu Lys Asn 85 90 95 Ala Ile Val Tyr AsnSer Met Tyr Glu Asn Phe Ser Thr Ser Phe Trp 100 105 110 Ile Arg Ile ProLys Tyr Phe Asn Ser Ile Ser Leu Asn Asn Glu Tyr 115 120 125 Thr Ile IleAsn Cys Met Glu Asn Asn Ser Gly Trp Lys Val Ser Leu 130 135 140 Asn TyrGly Glu Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu Ile Lys 145 150 155 160Gln Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn Ile Ser Asp Tyr 165 170175 Ile Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu Asn Asn 180185 190 Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro Ile Ser195 200 205 Asn Leu Gly Asn Ile His Ala Ser Asn Asn Ile Met Phe Lys LeuAsp 210 215 220 Gly Cys Arg Asp Thr His Arg Tyr Ile Trp Ile Lys Tyr PheAsn Leu 225 230 235 240 Phe Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys AspLeu Tyr Asp Asn 245 250 255 Gln Ser Asn Ser Gly Ile Leu Lys Asp Phe TrpGly Asp Tyr Leu Gln 260 265 270 Tyr Asp Lys Pro Tyr Tyr Met Leu Asn LeuTyr Asp Pro Asn Lys Tyr 275 280 285 Val Asp Val Asn Asn Val Gly Ile ArgGly Tyr Met Tyr Leu Lys Gly 290 295 300 Pro Arg Gly Ser Val Met Thr ThrAsn Ile Tyr Leu Asn Ser Ser Leu 305 310 315 320 Tyr Arg Gly Thr Lys PheIle Ile Lys Lys Tyr Ala Ser Gly Asn Lys 325 330 335 Asp Asn Ile Val ArgAsn Asn Asp Arg Val Tyr Ile Asn Val Val Val 340 345 350 Lys Asn Lys GluTyr Arg Leu Ala Thr Asn Ala Ser Gln Ala Gly Val 355 360 365 Glu Lys IleLeu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn Leu Ser 370 375 380 Gln ValVal Val Met Lys Ser Lys Asn Asp Gln Gly Ile Thr Asn Lys 385 390 395 400Cys Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly Phe Ile 405 410415 Gly Phe His Gln Phe Asn Asn Ile Ala Lys Leu Val Ala Ser Asn Trp 420425 430 Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr Leu Gly Cys Ser Trp435 440 445 Glu Phe Ile Pro Val Asp Asp Gly Trp Gly Glu Arg Pro Leu 450455 460 3891 base pairs nucleic acid double linear DNA (genomic) CDS1..3888 27 ATG CAA TTT GTT AAT AAA CAA TTT AAT TAT AAA GAT CCT GTA AATGGT 48 Met Gln Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly 15 10 15 GTT GAT ATT GCT TAT ATA AAA ATT CCA AAT GTA GGA CAA ATG CAA CCA96 Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Val Gly Gln Met Gln Pro 20 2530 GTA AAA GCT TTT AAA ATT CAT AAT AAA ATA TGG GTT ATT CCA GAA AGA 144Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45GAT ACA TTT ACA AAT CCT GAA GAA GGA GAT TTA AAT CCA CCA CCA GAA 192 AspThr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60 GCAAAA CAA GTT CCA GTT TCA TAT TAT GAT TCA ACA TAT TTA AGT ACA 240 Ala LysGln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr 65 70 75 80 GATAAT GAA AAA GAT AAT TAT TTA AAG GGA GTT ACA AAA TTA TTT GAG 288 Asp AsnGlu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95 AGA ATTTAT TCA ACT GAT CTT GGA AGA ATG TTG TTA ACA TCA ATA GTA 336 Arg Ile TyrSer Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110 AGG GGAATA CCA TTT TGG GGT GGA AGT ACA ATA GAT ACA GAA TTA AAA 384 Arg Gly IlePro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125 GTT ATTGAT ACT AAT TGT ATT AAT GTG ATA CAA CCA GAT GGT AGT TAT 432 Val Ile AspThr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135 140 AGA TCAGAA GAA CTT AAT CTA GTA ATA ATA GGA CCC TCA GCT GAT ATT 480 Arg Ser GluGlu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile 145 150 155 160 ATACAG TTT GAA TGT AAA AGC TTT GGA CAT GAA GTT TTG AAT CTT ACG 528 Ile GlnPhe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr 165 170 175 CGAAAT GGT TAT GGC TCT ACT CAA TAC ATT AGA TTT AGC CCA GAT TTT 576 Arg AsnGly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190 ACATTT GGT TTT GAG GAG TCA CTT GAA GTT GAT ACA AAT CCT CTT TTA 624 Thr PheGly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205 GGTGCA GGC AAA TTT GCT ACA GAT CCA GCA GTA ACA TTA GCA CAT GAA 672 Gly AlaGly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220 CTTATA CAT GCT GGA CAT AGA TTA TAT GGA ATA GCA ATT AAT CCA AAT 720 Leu IleHis Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn 225 230 235 240AGG GTT TTT AAA GTA AAT ACT AAT GCC TAT TAT GAA ATG AGT GGG TTA 768 ArgVal Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255GAA GTA AGC TTT GAG GAA CTT AGA ACA TTT GGG GGA CAT GAT GCA AAG 816 GluVal Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270TTT ATA GAT AGT TTA CAG GAA AAC GAA TTT CGT CTA TAT TAT TAT AAT 864 PheIle Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285AAG TTT AAA GAT ATA GCA AGT ACA CTT AAT AAA GCT AAA TCA ATA GTA 912 LysPhe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290 295 300GGT ACT ACT GCT TCA TTA CAG TAT ATG AAA AAT GTT TTT AAA GAG AAA 960 GlyThr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys 305 310 315320 TAT CTC CTA TCT GAA GAT ACA TCT GGA AAA TTT TCG GTA GAT AAA TTA 1008Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330335 AAA TTT GAT AAG TTA TAC AAA ATG TTA ACA GAG ATT TAC ACA GAG GAT 1056Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345350 AAT TTT GTT AAG TTT TTT AAA GTA CTT AAC AGA AAA ACA TAT TTG AAT 1104Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360365 TTT GAT AAA GCC GTA TTT AAG ATA AAT ATA GTA CCT AAG GTA AAT TAC 1152Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370 375380 ACA ATA TAT GAT GGA TTT AAT TTA AGA AAT ACA AAT TTA GCA GCA AAC 1200Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn 385 390395 400 TTT AAT GGT CAA AAT ACA GAA ATT AAT AAT ATG AAT TTT ACT AAA CTA1248 Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu 405410 415 AAA AAT TTT ACT GGA TTG TTT GAA TTT TAT AAG TTG CTA TGT GTA AGA1296 Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg 420425 430 GGG ATA ATA ACT TCT AAA ACT AAA TCA TTA GAT AAA GGA TAC AAT AAG1344 Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn Lys 435440 445 GCA TTA AAT GAT TTA TGT ATC AAA GTT AAT AAT TGG GAC TTG TTT TTT1392 Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe 450455 460 AGT CCT TCA GAA GAT AAT TTT ACT AAT GAT CTA AAT AAA GGA GAA GAA1440 Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu 465470 475 480 ATT ACA TCT GAT ACT AAT ATA GAA GCA GCA GAA GAA AAT ATT AGTTTA 1488 Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu485 490 495 GAT TTA ATA CAA CAA TAT TAT TTA ACC TTT AAT TTT GAT AAT GAACCT 1536 Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro500 505 510 GAA AAT ATT TCA ATA GAA AAT CTT TCA AGT GAC ATT ATA GGC CAATTA 1584 Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu515 520 525 GAA CTT ATG CCT AAT ATA GAA AGA TTT CCT AAT GGA AAA AAG TATGAG 1632 Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu530 535 540 TTA GAT AAA TAT ACT ATG TTC CAT TAT CTT CGT GCT CAA GAA TTTGAA 1680 Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu545 550 555 560 CAT GGT AAA TCT AGG ATT GCT TTA ACA AAT TCT GTT AAC GAAGCA TTA 1728 His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu AlaLeu 565 570 575 TTA AAT CCT AGT CGT GTT TAT ACA TTT TTT TCT TCA GAC TATGTA AAG 1776 Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr ValLys 580 585 590 AAA GTT AAT AAA GCT ACG GAG GCA GCT ATG TTT TTA GGC TGGGTA GAA 1824 Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp ValGlu 595 600 605 CAA TTA GTA TAT GAT TTT ACC GAT GAA ACT AGC GAA GTA AGTACT ACG 1872 Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser ThrThr 610 615 620 GAT AAA ATT GCG GAT ATA ACT ATA ATT ATT CCA TAT ATA GGACCT GCT 1920 Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly ProAla 625 630 635 640 TTA AAT ATA GGT AAT ATG TTA TAT AAA GAT GAT TTT GTAGGT GCT TTA 1968 Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val GlyAla Leu 645 650 655 ATA TTT TCA GGA GCT GTT ATT CTG TTA GAA TTT ATA CCAGAG ATT GCA 2016 Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro GluIle Ala 660 665 670 ATA CCT GTA TTA GGT ACT TTT GCA CTT GTA TCA TAT ATTGCG AAT AAG 2064 Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile AlaAsn Lys 675 680 685 GTT CTA ACC GTT CAA ACA ATA GAT AAT GCT TTA AGT AAAAGA AAT GAA 2112 Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys ArgAsn Glu 690 695 700 AAA TGG GAT GAG GTC TAT AAA TAT ATA GTA ACA AAT TGGTTA GCA AAG 2160 Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp LeuAla Lys 705 710 715 720 GTT AAT ACA CAG ATT GAT CTA ATA AGA AAA AAA ATGAAA GAA GCT TTA 2208 Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met LysGlu Ala Leu 725 730 735 GAA AAT CAA GCA GAA GCA ACA AAG GCT ATA ATA AACTAT CAG TAT AAT 2256 Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn TyrGln Tyr Asn 740 745 750 CAA TAT ACT GAG GAA GAG AAA AAT AAT ATT AAT TTTAAT ATT GAT GAT 2304 Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe AsnIle Asp Asp 755 760 765 TTA AGT TCG AAA CTT AAT GAG TCT ATA AAT AAA GCTATG ATT AAT ATA 2352 Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala MetIle Asn Ile 770 775 780 AAT AAA TTT TTG AAT CAA TGC TCT GTT TCA TAT TTAATG AAT TCT ATG 2400 Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu MetAsn Ser Met 785 790 795 800 ATC CCT TAT GGT GTT AAA CGG TTA GAA GAT TTTGAT GCT AGT CTT AAA 2448 Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe AspAla Ser Leu Lys 805 810 815 GAT GCA TTA TTA AAG TAT ATA TAT GAT AAT AGAGGA ACT TTA ATT GGT 2496 Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg GlyThr Leu Ile Gly 820 825 830 CAA GTA GAT AGA TTA AAA GAT AAA GTT AAT AATACA CTT AGT ACA GAT 2544 Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn ThrLeu Ser Thr Asp 835 840 845 ATA CCT TTT CAG CTT TCC AAA TAC GTA GAT AATCAA AGA TTA TTA TCT 2592 Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn GlnArg Leu Leu Ser 850 855 860 ACA TTT ACT GAA TAT ATT AAG AAT ATT ATT AATACT TCT ATA TTG AAT 2640 Thr Phe Thr Glu Tyr Ile Lys Asn Ile Ile Asn ThrSer Ile Leu Asn 865 870 875 880 TTA AGA TAT GAA AGT AAT CAT TTA ATA GACTTA TCT AGG TAT GCA TCA 2688 Leu Arg Tyr Glu Ser Asn His Leu Ile Asp LeuSer Arg Tyr Ala Ser 885 890 895 AAA ATA AAT ATT GGT AGT AAA GTA AAT TTTGAT CCA ATA GAT AAA AAT 2736 Lys Ile Asn Ile Gly Ser Lys Val Asn Phe AspPro Ile Asp Lys Asn 900 905 910 CAA ATT CAA TTA TTT AAT TTA GAA AGT AGTAAA ATT GAG GTA ATT TTA 2784 Gln Ile Gln Leu Phe Asn Leu Glu Ser Ser LysIle Glu Val Ile Leu 915 920 925 AAA AAT GCT ATT GTA TAT AAT AGT ATG TATGAA AAT TTT AGT ACT AGC 2832 Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr GluAsn Phe Ser Thr Ser 930 935 940 TTT TGG ATA AGA ATT CCT AAG TAT TTT AACAGT ATA AGT CTA AAT AAT 2880 Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn SerIle Ser Leu Asn Asn 945 950 955 960 GAA TAT ACA ATA ATA AAT TGT ATG GAAAAT AAT TCA GGA TGG AAA GTA 2928 Glu Tyr Thr Ile Ile Asn Cys Met Glu AsnAsn Ser Gly Trp Lys Val 965 970 975 TCA CTT AAT TAT GGT GAA ATA ATC TGGACT TTA CAG GAT ACT CAG GAA 2976 Ser Leu Asn Tyr Gly Glu Ile Ile Trp ThrLeu Gln Asp Thr Gln Glu 980 985 990 ATA AAA CAA AGA GTA GTT TTT AAA TACAGT CAA ATG ATT AAT ATA TCA 3024 Ile Lys Gln Arg Val Val Phe Lys Tyr SerGln Met Ile Asn Ile Ser 995 1000 1005 GAT TAT ATA AAC AGA TGG ATT TTTGTA ACT ATC ACT AAT AAT AGA TTA 3072 Asp Tyr Ile Asn Arg Trp Ile Phe ValThr Ile Thr Asn Asn Arg Leu 1010 1015 1020 AAT AAC TCT AAA ATT TAT ATAAAT GGA AGA TTA ATA GAT CAA AAA CCA 3120 Asn Asn Ser Lys Ile Tyr Ile AsnGly Arg Leu Ile Asp Gln Lys Pro 1025 1030 1035 1040 ATT TCA AAT TTA GGTAAT ATT CAT GCT AGT AAT AAT ATA ATG TTT AAA 3168 Ile Ser Asn Leu Gly AsnIle His Ala Ser Asn Asn Ile Met Phe Lys 1045 1050 1055 TTA GAT GGT TGTAGA GAT ACA CAT AGA TAT ATT TGG ATA AAA TAT TTT 3216 Leu Asp Gly Cys ArgAsp Thr His Arg Tyr Ile Trp Ile Lys Tyr Phe 1060 1065 1070 AAT CTT TTTGAT AAG GAA TTA AAT GAA AAA GAA ATC AAA GAT TTA TAT 3264 Asn Leu Phe AspLys Glu Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr 1075 1080 1085 GAT AATCAA TCA AAT TCA GGT ATT TTA AAA GAC TTT TGG GGT GAT TAT 3312 Asp Asn GlnSer Asn Ser Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr 1090 1095 1100 TTACAA TAT GAT AAA CCA TAC TAT ATG TTA AAT TTA TAT GAT CCA AAT 3360 Leu GlnTyr Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn 1105 1110 11151120 AAA TAT GTC GAT GTA AAT AAT GTA GGT ATT AGA GGT TAT ATG TAT CTT3408 Lys Tyr Val Asp Val Asn Asn Val Gly Ile Arg Gly Tyr Met Tyr Leu1125 1130 1135 AAA GGG CCT AGA GGT AGC GTA ATG ACT ACA AAC ATT TAT TTAAAT TCA 3456 Lys Gly Pro Arg Gly Ser Val Met Thr Thr Asn Ile Tyr Leu AsnSer 1140 1145 1150 AGT TTG TAT AGG GGG ACA AAA TTT ATT ATA AAA AAA TATGCT TCT GGA 3504 Ser Leu Tyr Arg Gly Thr Lys Phe Ile Ile Lys Lys Tyr AlaSer Gly 1155 1160 1165 AAT AAA GAT AAT ATT GTT AGA AAT AAT GAT CGT GTATAT ATT AAT GTA 3552 Asn Lys Asp Asn Ile Val Arg Asn Asn Asp Arg Val TyrIle Asn Val 1170 1175 1180 GTA GTT AAA AAT AAA GAA TAT AGG TTA GCT ACTAAT GCA TCA CAG GCA 3600 Val Val Lys Asn Lys Glu Tyr Arg Leu Ala Thr AsnAla Ser Gln Ala 1185 1190 1195 1200 GGC GTA GAA AAA ATA CTA AGT GCA TTAGAA ATA CCT GAT GTA GGA AAT 3648 Gly Val Glu Lys Ile Leu Ser Ala Leu GluIle Pro Asp Val Gly Asn 1205 1210 1215 CTA AGT CAA GTA GTA GTA ATG AAGTCA AAA AAT GAT CAA GGA ATA ACA 3696 Leu Ser Gln Val Val Val Met Lys SerLys Asn Asp Gln Gly Ile Thr 1220 1225 1230 AAT AAA TGC AAA ATG AAT TTACAA GAT AAT AAT GGG AAT GAT ATA GGC 3744 Asn Lys Cys Lys Met Asn Leu GlnAsp Asn Asn Gly Asn Asp Ile Gly 1235 1240 1245 TTT ATA GGA TTT CAT CAGTTT AAT AAT ATA GCT AAA CTA GTA GCA AGT 3792 Phe Ile Gly Phe His Gln PheAsn Asn Ile Ala Lys Leu Val Ala Ser 1250 1255 1260 AAT TGG TAT AAT AGACAA ATA GAA AGA TCT AGT AGG ACT TTG GGT TGC 3840 Asn Trp Tyr Asn Arg GlnIle Glu Arg Ser Ser Arg Thr Leu Gly Cys 1265 1270 1275 1280 TCA TGG GAATTT ATT CCT GTA GAT GAT GGA TGG GGA GAA AGG CCA CTG 3888 Ser Trp Glu PheIle Pro Val Asp Asp Gly Trp Gly Glu Arg Pro Leu 1285 1290 1295 TAA 38911296 amino acids amino acid linear protein 28 Met Gln Phe Val Asn LysGln Phe Asn Tyr Lys Asp Pro Val Asn Gly 1 5 10 15 Val Asp Ile Ala TyrIle Lys Ile Pro Asn Val Gly Gln Met Gln Pro 20 25 30 Val Lys Ala Phe LysIle His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45 Asp Thr Phe Thr AsnPro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60 Ala Lys Gln Val ProVal Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr 65 70 75 80 Asp Asn Glu LysAsp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95 Arg Ile Tyr SerThr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110 Arg Gly IlePro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125 Val IleAsp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135 140 ArgSer Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile 145 150 155160 Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr 165170 175 Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe180 185 190 Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro LeuLeu 195 200 205 Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu AlaHis Glu 210 215 220 Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala IleAsn Pro Asn 225 230 235 240 Arg Val Phe Lys Val Asn Thr Asn Ala Tyr TyrGlu Met Ser Gly Leu 245 250 255 Glu Val Ser Phe Glu Glu Leu Arg Thr PheGly Gly His Asp Ala Lys 260 265 270 Phe Ile Asp Ser Leu Gln Glu Asn GluPhe Arg Leu Tyr Tyr Tyr Asn 275 280 285 Lys Phe Lys Asp Ile Ala Ser ThrLeu Asn Lys Ala Lys Ser Ile Val 290 295 300 Gly Thr Thr Ala Ser Leu GlnTyr Met Lys Asn Val Phe Lys Glu Lys 305 310 315 320 Tyr Leu Leu Ser GluAsp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335 Lys Phe Asp LysLeu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350 Asn Phe ValLys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365 Phe AspLys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370 375 380 ThrIle Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn 385 390 395400 Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu 405410 415 Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg420 425 430 Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr AsnLys 435 440 445 Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp LeuPhe Phe 450 455 460 Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn LysGly Glu Glu 465 470 475 480 Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala GluGlu Asn Ile Ser Leu 485 490 495 Asp Leu Ile Gln Gln Tyr Tyr Leu Thr PheAsn Phe Asp Asn Glu Pro 500 505 510 Glu Asn Ile Ser Ile Glu Asn Leu SerSer Asp Ile Ile Gly Gln Leu 515 520 525 Glu Leu Met Pro Asn Ile Glu ArgPhe Pro Asn Gly Lys Lys Tyr Glu 530 535 540 Leu Asp Lys Tyr Thr Met PheHis Tyr Leu Arg Ala Gln Glu Phe Glu 545 550 555 560 His Gly Lys Ser ArgIle Ala Leu Thr Asn Ser Val Asn Glu Ala Leu 565 570 575 Leu Asn Pro SerArg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys 580 585 590 Lys Val AsnLys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu 595 600 605 Gln LeuVal Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr 610 615 620 AspLys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala 625 630 635640 Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu 645650 655 Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala660 665 670 Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala AsnLys 675 680 685 Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys ArgAsn Glu 690 695 700 Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn TrpLeu Ala Lys 705 710 715 720 Val Asn Thr Gln Ile Asp Leu Ile Arg Lys LysMet Lys Glu Ala Leu 725 730 735 Glu Asn Gln Ala Glu Ala Thr Lys Ala IleIle Asn Tyr Gln Tyr Asn 740 745 750 Gln Tyr Thr Glu Glu Glu Lys Asn AsnIle Asn Phe Asn Ile Asp Asp 755 760 765 Leu Ser Ser Lys Leu Asn Glu SerIle Asn Lys Ala Met Ile Asn Ile 770 775 780 Asn Lys Phe Leu Asn Gln CysSer Val Ser Tyr Leu Met Asn Ser Met 785 790 795 800 Ile Pro Tyr Gly ValLys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys 805 810 815 Asp Ala Leu LeuLys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly 820 825 830 Gln Val AspArg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp 835 840 845 Ile ProPhe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser 850 855 860 ThrPhe Thr Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn 865 870 875880 Leu Arg Tyr Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser 885890 895 Lys Ile Asn Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn900 905 910 Gln Ile Gln Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val IleLeu 915 920 925 Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe SerThr Ser 930 935 940 Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile SerLeu Asn Asn 945 950 955 960 Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn AsnSer Gly Trp Lys Val 965 970 975 Ser Leu Asn Tyr Gly Glu Ile Ile Trp ThrLeu Gln Asp Thr Gln Glu 980 985 990 Ile Lys Gln Arg Val Val Phe Lys TyrSer Gln Met Ile Asn Ile Ser 995 1000 1005 Asp Tyr Ile Asn Arg Trp IlePhe Val Thr Ile Thr Asn Asn Arg Leu 1010 1015 1020 Asn Asn Ser Lys IleTyr Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro 1025 1030 1035 1040 Ile SerAsn Leu Gly Asn Ile His Ala Ser Asn Asn Ile Met Phe Lys 1045 1050 1055Leu Asp Gly Cys Arg Asp Thr His Arg Tyr Ile Trp Ile Lys Tyr Phe 10601065 1070 Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp LeuTyr 1075 1080 1085 Asp Asn Gln Ser Asn Ser Gly Ile Leu Lys Asp Phe TrpGly Asp Tyr 1090 1095 1100 Leu Gln Tyr Asp Lys Pro Tyr Tyr Met Leu AsnLeu Tyr Asp Pro Asn 1105 1110 1115 1120 Lys Tyr Val Asp Val Asn Asn ValGly Ile Arg Gly Tyr Met Tyr Leu 1125 1130 1135 Lys Gly Pro Arg Gly SerVal Met Thr Thr Asn Ile Tyr Leu Asn Ser 1140 1145 1150 Ser Leu Tyr ArgGly Thr Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly 1155 1160 1165 Asn LysAsp Asn Ile Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val 1170 1175 1180Val Val Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala 11851190 1195 1200 Gly Val Glu Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp ValGly Asn 1205 1210 1215 Leu Ser Gln Val Val Val Met Lys Ser Lys Asn AspGln Gly Ile Thr 1220 1225 1230 Asn Lys Cys Lys Met Asn Leu Gln Asp AsnAsn Gly Asn Asp Ile Gly 1235 1240 1245 Phe Ile Gly Phe His Gln Phe AsnAsn Ile Ala Lys Leu Val Ala Ser 1250 1255 1260 Asn Trp Tyr Asn Arg GlnIle Glu Arg Ser Ser Arg Thr Leu Gly Cys 1265 1270 1275 1280 Ser Trp GluPhe Ile Pro Val Asp Asp Gly Trp Gly Glu Arg Pro Leu 1285 1290 1295 30base pairs nucleic acid single linear other nucleic acid /desc = “DNA”29 CGCCATGGCT AGATTATTAT CTACATTTAC 30 26 base pairs nucleic acid singlelinear other nucleic acid /desc = “DNA” 30 GCAAGCTTCT TGACAGACTC ATGTAG26 1546 base pairs nucleic acid double linear DNA (genomic) 31AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACCATG GGCCATCATC 120ATCATCATCA TCATCATCAT CACAGCAGCG GCCATATCGA AGGTCGTCAT ATGGCTAGCA 180TGGCTAGATT ATTATCTACA TTTACTGAAT ATATTAAGAA TATTATTAAT ACTTCTATAT 240TGAATTTAAG ATATGAAAGT AATCATTTAA TAGACTTATC TAGGTATGCA TCAAAAATAA 300ATATTGGTAG TAAAGTAAAT TTTGATCCAA TAGATAAAAA TCAAATTCAA TTATTTAATT 360TAGAAAGTAG TAAAATTGAG GTAATTTTAA AAAATGCTAT TGTATATAAT AGTATGTATG 420AAAATTTTAG TACTAGCTTT TGGATAAGAA TTCCTAAGTA TTTTAACAGT ATAAGTCTAA 480ATAATGAATA TACAATAATA AATTGTATGG AAAATAATTC AGGATGGAAA GTATCACTTA 540ATTATGGTGA AATAATCTGG ACTTTACAGG ATACTCAGGA AATAAAACAA AGAGTAGTTT 600TTAAATACAG TCAAATGATT AATATATCAG ATTATATAAA CAGATGGATT TTTGTAACTA 660TCACTAATAA TAGATTAAAT AACTCTAAAA TTTATATAAA TGGAAGATTA ATAGATCAAA 720AACCAATTTC AAATTTAGGT AATATTCATG CTAGTAATAA TATAATGTTT AAATTAGATG 780GTTGTAGAGA TACACATAGA TATATTTGGA TAAAATATTT TAATCTTTTT GATAAGGAAT 840TAAATGAAAA AGAAATCAAA GATTTATATG ATAATCAATC AAATTCAGGT ATTTTAAAAG 900ACTTTTGGGG TGATTATTTA CAATATGATA AACCATACTA TATGTTAAAT TTATATGATC 960CAAATAAATA TGTCGATGTA AATAATGTAG GTATTAGAGG TTATATGTAT CTTAAAGGGC 1020CTAGAGGTAG CGTAATGACT ACAAACATTT ATTTAAATTC AAGTTTGTAT AGGGGGACAA 1080AATTTATTAT AAAAAAATAT GCTTCTGGAA ATAAAGATAA TATTGTTAGA AATAATGATC 1140GTGTATATAT TAATGTAGTA GTTAAAAATA AAGAATATAG GTTAGCTACT AATGCATCAC 1200AGGCAGGCGT AGAAAAAATA CTAAGTGCAT TAGAAATACC TGATGTAGGA AATCTAAGTC 1260AAGTAGTAGT AATGAAGTCA AAAAATGATC AAGGAATAAC AAATAAATGC AAAATGAATT 1320TACAAGATAA TAATGGGAAT GATATAGGCT TTATAGGATT TCATCAGTTT AATAATATAG 1380CTAAACTAGT AGCAAGTAAT TGGTATAATA GACAAATAGA AAGATCTAGT AGGACTTTGG 1440GTTGCTCATG GGAATTTATT CCTGTAGATG ATGGATGGGG AGAAAGGCCA CTGTAATTAA 1500TCTCAAACTA CATGAGTCTG TCAAGAAGCT TGCGGCCGCA CTCGAG 1546 9 amino acidsamino acid Not Relevant Not Relevant peptide 32 Met His His His His HisHis Met Ala 1 5 21 base pairs nucleic acid single linear other nucleicacid /desc = “DNA” 33 TATGCATCAC CATCACCATC A 21 23 base pairs nucleicacid single linear other nucleic acid /desc = “DNA” 34 CATGTGATGGTGATGGTGAT GCA 23 1351 base pairs nucleic acid double linear othernucleic acid /desc = “DNA” CDS 1..1335 35 ATG CAT CAC CAT CAC CAT CACATG GCT CGT CTG CTG TCT ACC TTC ACT 48 Met His His His His His His MetAla Arg Leu Leu Ser Thr Phe Thr 1 5 10 15 GAA TAC ATC AAG AAC ATC ATCAAT ACC TCC ATC CTG AAC CTG CGC TAC 96 Glu Tyr Ile Lys Asn Ile Ile AsnThr Ser Ile Leu Asn Leu Arg Tyr 20 25 30 GAA TCC AAT CAC CTG ATC GAC CTGTCT CGC TAC GCT TCC AAA ATC AAC 144 Glu Ser Asn His Leu Ile Asp Leu SerArg Tyr Ala Ser Lys Ile Asn 35 40 45 ATC GGT TCT AAA GTT AAC TTC GAT CCGATC GAC AAG AAT CAG ATC CAG 192 Ile Gly Ser Lys Val Asn Phe Asp Pro IleAsp Lys Asn Gln Ile Gln 50 55 60 CTG TTC AAT CTG GAA TCT TCC AAA ATC GAAGTT ATC CTG AAG AAT GCT 240 Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu ValIle Leu Lys Asn Ala 65 70 75 80 ATC GTA TAC AAC TCT ATG TAC GAA AAC TTCTCC ACC TCC TTC TGG ATC 288 Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe SerThr Ser Phe Trp Ile 85 90 95 CGT ATC CCG AAA TAC TTC AAC TCC ATC TCT CTGAAC AAT GAA TAC ACC 336 Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu AsnAsn Glu Tyr Thr 100 105 110 ATC ATC AAC TGC ATG GAA AAC AAT TCT GGT TGGAAA GTA TCT CTG AAC 384 Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp LysVal Ser Leu Asn 115 120 125 TAC GGT GAA ATC ATC TGG ACT CTG CAG GAC ACTCAG GAA ATC AAA CAG 432 Tyr Gly Glu Ile Ile Trp Thr Leu Gln Asp Thr GlnGlu Ile Lys Gln 130 135 140 CGT GTT GTA TTC AAA TAC TCT CAG ATG ATC AACATC TCT GAC TAC ATC 480 Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn IleSer Asp Tyr Ile 145 150 155 160 AAT CGC TGG ATC TTC GTT ACC ATC ACC AACAAT CGT CTG AAT AAC TCC 528 Asn Arg Trp Ile Phe Val Thr Ile Thr Asn AsnArg Leu Asn Asn Ser 165 170 175 AAA ATC TAC ATC AAC GGC CGT CTG ATC GACCAG AAA CCG ATC TCC AAT 576 Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp GlnLys Pro Ile Ser Asn 180 185 190 CTG GGT AAC ATC CAC GCT TCT AAT AAC ATCATG TTC AAA CTG GAC GGT 624 Leu Gly Asn Ile His Ala Ser Asn Asn Ile MetPhe Lys Leu Asp Gly 195 200 205 TGT CGT GAC ACT CAC CGC TAC ATC TGG ATCAAA TAC TTC AAT CTG TTC 672 Cys Arg Asp Thr His Arg Tyr Ile Trp Ile LysTyr Phe Asn Leu Phe 210 215 220 GAC AAA GAA CTG AAC GAA AAA GAA ATC AAAGAC CTG TAC GAC AAC CAG 720 Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys AspLeu Tyr Asp Asn Gln 225 230 235 240 TCC AAT TCT GGT ATC CTG AAA GAC TTCTGG GGT GAC TAC CTG CAG TAC 768 Ser Asn Ser Gly Ile Leu Lys Asp Phe TrpGly Asp Tyr Leu Gln Tyr 245 250 255 GAC AAA CCG TAC TAC ATG CTG AAT CTGTAC GAT CCG AAC AAA TAC GTT 816 Asp Lys Pro Tyr Tyr Met Leu Asn Leu TyrAsp Pro Asn Lys Tyr Val 260 265 270 GAC GTC AAC AAT GTA GGT ATC CGC GGTTAC ATG TAC CTG AAA GGT CCG 864 Asp Val Asn Asn Val Gly Ile Arg Gly TyrMet Tyr Leu Lys Gly Pro 275 280 285 CGT GGT TCT GTT ATG ACT ACC AAC ATCTAC CTG AAC TCT TCC CTG TAC 912 Arg Gly Ser Val Met Thr Thr Asn Ile TyrLeu Asn Ser Ser Leu Tyr 290 295 300 CGT GGT ACC AAA TTC ATC ATC AAG AAATAC GCG TCT GGT AAC AAG GAC 960 Arg Gly Thr Lys Phe Ile Ile Lys Lys TyrAla Ser Gly Asn Lys Asp 305 310 315 320 AAT ATC GTT CGC AAC AAT GAT CGTGTA TAC ATC AAT GTT GTA GTT AAG 1008 Asn Ile Val Arg Asn Asn Asp Arg ValTyr Ile Asn Val Val Val Lys 325 330 335 AAC AAA GAA TAC CGT CTG GCT ACCAAT GCT TCT CAG GCT GGT GTA GAA 1056 Asn Lys Glu Tyr Arg Leu Ala Thr AsnAla Ser Gln Ala Gly Val Glu 340 345 350 AAG ATC TTG TCT GCT CTG GAA ATCCCG GAC GTT GGT AAT CTG TCT CAG 1104 Lys Ile Leu Ser Ala Leu Glu Ile ProAsp Val Gly Asn Leu Ser Gln 355 360 365 GTA GTT GTA ATG AAA TCC AAG AACGAC CAG GGT ATC ACT AAC AAA TGC 1152 Val Val Val Met Lys Ser Lys Asn AspGln Gly Ile Thr Asn Lys Cys 370 375 380 AAA ATG AAT CTG CAG GAC AAC AATGGT AAC GAT ATC GGT TTC ATC GGT 1200 Lys Met Asn Leu Gln Asp Asn Asn GlyAsn Asp Ile Gly Phe Ile Gly 385 390 395 400 TTC CAC CAG TTC AAC AAT ATCGCT AAA CTG GTT GCT TCC AAC TGG TAC 1248 Phe His Gln Phe Asn Asn Ile AlaLys Leu Val Ala Ser Asn Trp Tyr 405 410 415 AAT CGT CAG ATC GAA CGT TCCTCT CGC ACT CTG GGT TGC TCT TGG GAG 1296 Asn Arg Gln Ile Glu Arg Ser SerArg Thr Leu Gly Cys Ser Trp Glu 420 425 430 TTC ATC CCG GTT GAT GAC GGTTGG GGT GAA CGT CCG CTG TAACCCGGGA 1345 Phe Ile Pro Val Asp Asp Gly TrpGly Glu Arg Pro Leu 435 440 445 AAGCTT 1351 445 amino acids amino acidlinear protein 36 Met His His His His His His Met Ala Arg Leu Leu SerThr Phe Thr 1 5 10 15 Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile LeuAsn Leu Arg Tyr 20 25 30 Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr AlaSer Lys Ile Asn 35 40 45 Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp LysAsn Gln Ile Gln 50 55 60 Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val IleLeu Lys Asn Ala 65 70 75 80 Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe SerThr Ser Phe Trp Ile 85 90 95 Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser LeuAsn Asn Glu Tyr Thr 100 105 110 Ile Ile Asn Cys Met Glu Asn Asn Ser GlyTrp Lys Val Ser Leu Asn 115 120 125 Tyr Gly Glu Ile Ile Trp Thr Leu GlnAsp Thr Gln Glu Ile Lys Gln 130 135 140 Arg Val Val Phe Lys Tyr Ser GlnMet Ile Asn Ile Ser Asp Tyr Ile 145 150 155 160 Asn Arg Trp Ile Phe ValThr Ile Thr Asn Asn Arg Leu Asn Asn Ser 165 170 175 Lys Ile Tyr Ile AsnGly Arg Leu Ile Asp Gln Lys Pro Ile Ser Asn 180 185 190 Leu Gly Asn IleHis Ala Ser Asn Asn Ile Met Phe Lys Leu Asp Gly 195 200 205 Cys Arg AspThr His Arg Tyr Ile Trp Ile Lys Tyr Phe Asn Leu Phe 210 215 220 Asp LysGlu Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr Asp Asn Gln 225 230 235 240Ser Asn Ser Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr Leu Gln Tyr 245 250255 Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn Lys Tyr Val 260265 270 Asp Val Asn Asn Val Gly Ile Arg Gly Tyr Met Tyr Leu Lys Gly Pro275 280 285 Arg Gly Ser Val Met Thr Thr Asn Ile Tyr Leu Asn Ser Ser LeuTyr 290 295 300 Arg Gly Thr Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly AsnLys Asp 305 310 315 320 Asn Ile Val Arg Asn Asn Asp Arg Val Tyr Ile AsnVal Val Val Lys 325 330 335 Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala SerGln Ala Gly Val Glu 340 345 350 Lys Ile Leu Ser Ala Leu Glu Ile Pro AspVal Gly Asn Leu Ser Gln 355 360 365 Val Val Val Met Lys Ser Lys Asn AspGln Gly Ile Thr Asn Lys Cys 370 375 380 Lys Met Asn Leu Gln Asp Asn AsnGly Asn Asp Ile Gly Phe Ile Gly 385 390 395 400 Phe His Gln Phe Asn AsnIle Ala Lys Leu Val Ala Ser Asn Trp Tyr 405 410 415 Asn Arg Gln Ile GluArg Ser Ser Arg Thr Leu Gly Cys Ser Trp Glu 420 425 430 Phe Ile Pro ValAsp Asp Gly Trp Gly Glu Arg Pro Leu 435 440 445 27 base pairs nucleicacid single linear other nucleic acid /desc = “DNA” 37 CGCATATGAATATTCGTCCA TTGCATG 27 27 base pairs nucleic acid single linear othernucleic acid /desc = “DNA” 38 GGAAGCTTGC AGGGCAATTA CATCATG 27 3876 basepairs nucleic acid double linear DNA (genomic) CDS 1..3873 39 ATG CCAGTT ACA ATA AAT AAT TTT AAT TAT AAT GAT CCT ATT GAT AAT 48 Met Pro ValThr Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn 1 5 10 15 GAC AATATT ATT ATG ATG GAA CCT CCA TTT GCA AGG GGT ACG GGG AGA 96 Asp Asn IleIle Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg 20 25 30 TAT TAT AAAGCT TTT AAA ATC ACA GAT CGT ATT TGG ATA ATA CCC GAA 144 Tyr Tyr Lys AlaPhe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu 35 40 45 AGA TAT ACT TTTGGA TAT AAA CCT GAG GAT TTT AAT AAA AGT TCC GGT 192 Arg Tyr Thr Phe GlyTyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly 50 55 60 ATT TTT AAT AGA GATGTT TGT GAA TAT TAT GAT CCA GAT TAC TTA AAT 240 Ile Phe Asn Arg Asp ValCys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn 65 70 75 80 ACC AAT GAT AAA AAGAAT ATA TTT TTC CAA ACA TTG ATC AAG TTA TTT 288 Thr Asn Asp Lys Lys AsnIle Phe Phe Gln Thr Leu Ile Lys Leu Phe 85 90 95 AAT AGA ATC AAA TCA AAACCA TTG GGT GAA AAG TTA TTA GAG ATG ATT 336 Asn Arg Ile Lys Ser Lys ProLeu Gly Glu Lys Leu Leu Glu Met Ile 100 105 110 ATA AAT GGT ATA CCT TATCTT GGA GAT AGA CGT GTT CCA CTC GAA GAG 384 Ile Asn Gly Ile Pro Tyr LeuGly Asp Arg Arg Val Pro Leu Glu Glu 115 120 125 TTT AAC ACA AAC ATT GCTAGT GTA ACT GTT AAT AAA TTA ATT AGT AAT 432 Phe Asn Thr Asn Ile Ala SerVal Thr Val Asn Lys Leu Ile Ser Asn 130 135 140 CCA GGA GAA GTG GAG CGAAAA AAA GGT ATT TTC GCA AAT TTA ATA ATA 480 Pro Gly Glu Val Glu Arg LysLys Gly Ile Phe Ala Asn Leu Ile Ile 145 150 155 160 TTT GGA CCT GGG CCAGTT TTA AAT GAA AAT GAG ACT ATA GAT ATA GGT 528 Phe Gly Pro Gly Pro ValLeu Asn Glu Asn Glu Thr Ile Asp Ile Gly 165 170 175 ATA CAA AAT CAT TTTGCA TCA AGG GAA GGC TTT GGG GGT ATA ATG CAA 576 Ile Gln Asn His Phe AlaSer Arg Glu Gly Phe Gly Gly Ile Met Gln 180 185 190 ATG AAA TTT TGT CCAGAA TAT GTA AGC GTA TTT AAT AAT GTT CAA GAA 624 Met Lys Phe Cys Pro GluTyr Val Ser Val Phe Asn Asn Val Gln Glu 195 200 205 AAC AAA GGC GCA AGTATA TTT AAT AGA CGT GGA TAT TTT TCA GAT CCA 672 Asn Lys Gly Ala Ser IlePhe Asn Arg Arg Gly Tyr Phe Ser Asp Pro 210 215 220 GCC TTG ATA TTA ATGCAT GAA CTT ATA CAT GTT TTG CAT GGA TTA TAT 720 Ala Leu Ile Leu Met HisGlu Leu Ile His Val Leu His Gly Leu Tyr 225 230 235 240 GGC ATT AAA GTAGAT GAT TTA CCA ATT GTA CCA AAT GAA AAA AAA TTT 768 Gly Ile Lys Val AspAsp Leu Pro Ile Val Pro Asn Glu Lys Lys Phe 245 250 255 TTT ATG CAA TCTACA GAT ACT ATA CAG GCA GAA GAA CTA TAT ACA TTT 816 Phe Met Gln Ser ThrAsp Thr Ile Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270 GGA GGA CAA GATCCC AGC ATC ATA TCT CCT TCT ACA GAT AAA AGT ATC 864 Gly Gly Gln Asp ProSer Ile Ile Ser Pro Ser Thr Asp Lys Ser Ile 275 280 285 TAT GAT AAA GTTTTG CAA AAT TTT AGG GGG ATA GTT GAT AGA CTT AAC 912 Tyr Asp Lys Val LeuGln Asn Phe Arg Gly Ile Val Asp Arg Leu Asn 290 295 300 AAG GTT TTA GTTTGC ATA TCA GAT CCT AAC ATT AAC ATT AAT ATA TAT 960 Lys Val Leu Val CysIle Ser Asp Pro Asn Ile Asn Ile Asn Ile Tyr 305 310 315 320 AAA AAT AAATTT AAA GAT AAA TAT AAA TTC GTT GAA GAT TCT GAA GGA 1008 Lys Asn Lys PheLys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly 325 330 335 AAA TAT AGTATA GAT GTA GAA AGT TTC AAT AAA TTA TAT AAA AGC TTA 1056 Lys Tyr Ser IleAsp Val Glu Ser Phe Asn Lys Leu Tyr Lys Ser Leu 340 345 350 ATG TTA GGTTTT ACA GAA ATT AAT ATA GCA GAA AAT TAT AAA ATA AAA 1104 Met Leu Gly PheThr Glu Ile Asn Ile Ala Glu Asn Tyr Lys Ile Lys 355 360 365 ACT AGA GCTTCT TAT TTT AGT GAT TCC TTA CCA CCA GTA AAA ATA AAA 1152 Thr Arg Ala SerTyr Phe Ser Asp Ser Leu Pro Pro Val Lys Ile Lys 370 375 380 AAT TTA TTAGAT AAT GAA ATC TAT ACT ATA GAG GAA GGG TTT AAT ATA 1200 Asn Leu Leu AspAsn Glu Ile Tyr Thr Ile Glu Glu Gly Phe Asn Ile 385 390 395 400 TCT GATAAA AAT ATG GGA AAA GAA TAT AGG GGT CAG AAT AAA GCT ATA 1248 Ser Asp LysAsn Met Gly Lys Glu Tyr Arg Gly Gln Asn Lys Ala Ile 405 410 415 AAT AAACAA GCT TAT GAA GAA ATC AGC AAG GAG CAT TTG GCT GTA TAT 1296 Asn Lys GlnAla Tyr Glu Glu Ile Ser Lys Glu His Leu Ala Val Tyr 420 425 430 AAG ATACAA ATG TGT AAA AGT GTT AAA GTT CCA GGA ATA TGT ATT GAT 1344 Lys Ile GlnMet Cys Lys Ser Val Lys Val Pro Gly Ile Cys Ile Asp 435 440 445 GTC GATAAT GAA AAT TTG TTC TTT ATA GCT GAT AAA AAT AGT TTT TCA 1392 Val Asp AsnGlu Asn Leu Phe Phe Ile Ala Asp Lys Asn Ser Phe Ser 450 455 460 GAT GATTTA TCT AAA AAT GAA AGA GTA GAA TAT AAT ACA CAG AAT AAT 1440 Asp Asp LeuSer Lys Asn Glu Arg Val Glu Tyr Asn Thr Gln Asn Asn 465 470 475 480 TATATA GGA AAT GAC TTT CCT ATA AAT GAA TTA ATT TTA GAT ACT GAT 1488 Tyr IleGly Asn Asp Phe Pro Ile Asn Glu Leu Ile Leu Asp Thr Asp 485 490 495 TTAATA AGT AAA ATA GAA TTA CCA AGT GAA AAT ACA GAA TCA CTT ACT 1536 Leu IleSer Lys Ile Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr 500 505 510 GATTTT AAT GTA GAT GTT CCA GTA TAT GAA AAA CAA CCC GCT ATA AAA 1584 Asp PheAsn Val Asp Val Pro Val Tyr Glu Lys Gln Pro Ala Ile Lys 515 520 525 AAAGTT TTT ACA GAT GAA AAT ACC ATC TTT CAA TAT TTA TAC TCT CAG 1632 Lys ValPhe Thr Asp Glu Asn Thr Ile Phe Gln Tyr Leu Tyr Ser Gln 530 535 540 ACATTT CCT CTA AAT ATA AGA GAT ATA AGT TTA ACA TCT TCA TTT GAT 1680 Thr PhePro Leu Asn Ile Arg Asp Ile Ser Leu Thr Ser Ser Phe Asp 545 550 555 560GAT GCA TTA TTA GTT TCT AGC AAA GTT TAT TCA TTT TTT TCT ATG GAT 1728 AspAla Leu Leu Val Ser Ser Lys Val Tyr Ser Phe Phe Ser Met Asp 565 570 575TAT ATT AAA ACT GCT AAT AAA GTA GTA GAA GCA GGA TTA TTT GCA GGT 1776 TyrIle Lys Thr Ala Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly 580 585 590TGG GTG AAA CAG ATA GTA GAT GAT TTT GTA ATC GAA GCT AAT AAA AGC 1824 TrpVal Lys Gln Ile Val Asp Asp Phe Val Ile Glu Ala Asn Lys Ser 595 600 605AGT ACT ATG GAT AAA ATT GCA GAT ATA TCT CTA ATT GTT CCT TAT ATA 1872 SerThr Met Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Ile 610 615 620GGA TTA GCT TTA AAT GTA GGA GAT GAA ACA GCT AAA GGA AAT TTT GAA 1920 GlyLeu Ala Leu Asn Val Gly Asp Glu Thr Ala Lys Gly Asn Phe Glu 625 630 635640 AGT GCT TTT GAG ATT GCA GGA TCC AGT ATT TTA CTA GAA TTT ATA CCA 1968Ser Ala Phe Glu Ile Ala Gly Ser Ser Ile Leu Leu Glu Phe Ile Pro 645 650655 GAA CTT TTA ATA CCT GTA GTT GGA GTC TTT TTA TTA GAA TCA TAT ATT 2016Glu Leu Leu Ile Pro Val Val Gly Val Phe Leu Leu Glu Ser Tyr Ile 660 665670 GAC AAT AAA AAT AAA ATT ATT AAA ACA ATA GAT AAT GCT TTA ACT AAA 2064Asp Asn Lys Asn Lys Ile Ile Lys Thr Ile Asp Asn Ala Leu Thr Lys 675 680685 AGA GTG GAA AAA TGG ATT GAT ATG TAC GGA TTA ATA GTA GCG CAA TGG 2112Arg Val Glu Lys Trp Ile Asp Met Tyr Gly Leu Ile Val Ala Gln Trp 690 695700 CTC TCA ACA GTT AAT ACT CAA TTT TAT ACA ATA AAA GAG GGA ATG TAT 2160Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile Lys Glu Gly Met Tyr 705 710715 720 AAG GCT TTA AAT TAT CAA GCA CAA GCA TTG GAA GAA ATA ATA AAA TAC2208 Lys Ala Leu Asn Tyr Gln Ala Gln Ala Leu Glu Glu Ile Ile Lys Tyr 725730 735 AAA TAT AAT ATA TAT TCT GAA GAG GAA AAG TCA AAT ATT AAC ATC AAT2256 Lys Tyr Asn Ile Tyr Ser Glu Glu Glu Lys Ser Asn Ile Asn Ile Asn 740745 750 TTT AAT GAT ATA AAT TCT AAA CTT AAT GAT GGT ATT AAC CAA GCT ATG2304 Phe Asn Asp Ile Asn Ser Lys Leu Asn Asp Gly Ile Asn Gln Ala Met 755760 765 GAT AAT ATA AAT GAT TTT ATA AAT GAA TGT TCT GTA TCA TAT TTA ATG2352 Asp Asn Ile Asn Asp Phe Ile Asn Glu Cys Ser Val Ser Tyr Leu Met 770775 780 AAA AAA ATG ATT CCA TTA GCT GTA AAA AAA TTA CTA GAC TTT GAT AAT2400 Lys Lys Met Ile Pro Leu Ala Val Lys Lys Leu Leu Asp Phe Asp Asn 785790 795 800 ACT CTC AAA AAA AAT TTA TTA AAT TAT ATA GAT GAA AAT AAA TTATAT 2448 Thr Leu Lys Lys Asn Leu Leu Asn Tyr Ile Asp Glu Asn Lys Leu Tyr805 810 815 TTA ATT GGA AGT GTA GAA GAT GAA AAA TCA AAA GTA GAT AAA TACTTG 2496 Leu Ile Gly Ser Val Glu Asp Glu Lys Ser Lys Val Asp Lys Tyr Leu820 825 830 AAA ACC ATT ATA CCA TTT GAT CTT TCA ACG TAT TCT AAT ATT GAAATA 2544 Lys Thr Ile Ile Pro Phe Asp Leu Ser Thr Tyr Ser Asn Ile Glu Ile835 840 845 CTA ATA AAA ATA TTT AAT AAA TAT AAT AGC GAA ATT TTA AAT AATATT 2592 Leu Ile Lys Ile Phe Asn Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile850 855 860 ATC TTA AAT TTA AGA TAT AGA GAT AAT AAT TTA ATA GAT TTA TCAGGA 2640 Ile Leu Asn Leu Arg Tyr Arg Asp Asn Asn Leu Ile Asp Leu Ser Gly865 870 875 880 TAT GGA GCA AAG GTA GAG GTA TAT GAT GGG GTC AAG CTT AATGAT AAA 2688 Tyr Gly Ala Lys Val Glu Val Tyr Asp Gly Val Lys Leu Asn AspLys 885 890 895 AAT CAA TTT AAA TTA ACT AGT TCA GCA GAT AGT AAG ATT AGAGTC ACT 2736 Asn Gln Phe Lys Leu Thr Ser Ser Ala Asp Ser Lys Ile Arg ValThr 900 905 910 CAA AAT CAG AAT ATT ATA TTT AAT AGT ATG TTC CTT GAT TTTAGC GTT 2784 Gln Asn Gln Asn Ile Ile Phe Asn Ser Met Phe Leu Asp Phe SerVal 915 920 925 AGC TTT TGG ATA AGG ATA CCT AAA TAT AGG AAT GAT GAT ATACAA AAT 2832 Ser Phe Trp Ile Arg Ile Pro Lys Tyr Arg Asn Asp Asp Ile GlnAsn 930 935 940 TAT ATT CAT AAT GAA TAT ACG ATA ATT AAT TGT ATG AAA AATAAT TCA 2880 Tyr Ile His Asn Glu Tyr Thr Ile Ile Asn Cys Met Lys Asn AsnSer 945 950 955 960 GGC TGG AAA ATA TCT ATT AGG GGT AAT AGG ATA ATA TGGACC TTA ATT 2928 Gly Trp Lys Ile Ser Ile Arg Gly Asn Arg Ile Ile Trp ThrLeu Ile 965 970 975 GAT ATA AAT GGA AAA ACC AAA TCA GTA TTT TTT GAA TATAAC ATA AGA 2976 Asp Ile Asn Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr AsnIle Arg 980 985 990 GAA GAT ATA TCA GAG TAT ATA AAT AGA TGG TTT TTT GTAACT ATT ACT 3024 Glu Asp Ile Ser Glu Tyr Ile Asn Arg Trp Phe Phe Val ThrIle Thr 995 1000 1005 AAT AAT TTG GAT AAT GCT AAA ATT TAT ATT AAT GGCACG TTA GAA TCA 3072 Asn Asn Leu Asp Asn Ala Lys Ile Tyr Ile Asn Gly ThrLeu Glu Ser 1010 1015 1020 AAT ATG GAT ATT AAA GAT ATA GGA GAA GTT ATTGTT AAT GGT GAA ATA 3120 Asn Met Asp Ile Lys Asp Ile Gly Glu Val Ile ValAsn Gly Glu Ile 1025 1030 1035 1040 ACA TTT AAA TTA GAT GGT GAT GTA GATAGA ACA CAA TTT ATT TGG ATG 3168 Thr Phe Lys Leu Asp Gly Asp Val Asp ArgThr Gln Phe Ile Trp Met 1045 1050 1055 AAA TAT TTT AGT ATT TTT AAT ACGCAA TTA AAT CAA TCA AAT ATT AAA 3216 Lys Tyr Phe Ser Ile Phe Asn Thr GlnLeu Asn Gln Ser Asn Ile Lys 1060 1065 1070 GAG ATA TAT AAA ATT CAA TCATAT AGC GAA TAC TTA AAA GAT TTT TGG 3264 Glu Ile Tyr Lys Ile Gln Ser TyrSer Glu Tyr Leu Lys Asp Phe Trp 1075 1080 1085 GGA AAT CCT TTA ATG TATAAT AAA GAA TAT TAT ATG TTT AAT GCG GGG 3312 Gly Asn Pro Leu Met Tyr AsnLys Glu Tyr Tyr Met Phe Asn Ala Gly 1090 1095 1100 AAT AAA AAT TCA TATATT AAA CTA GTG AAA GAT TCA TCT GTA GGT GAA 3360 Asn Lys Asn Ser Tyr IleLys Leu Val Lys Asp Ser Ser Val Gly Glu 1105 1110 1115 1120 ATA TTA ATACGT AGC AAA TAT AAT CAG AAT TCC AAT TAT ATA AAT TAT 3408 Ile Leu Ile ArgSer Lys Tyr Asn Gln Asn Ser Asn Tyr Ile Asn Tyr 1125 1130 1135 AGA AATTTA TAT ATT GGA GAA AAA TTT ATT ATA AGA AGA GAG TCA AAT 3456 Arg Asn LeuTyr Ile Gly Glu Lys Phe Ile Ile Arg Arg Glu Ser Asn 1140 1145 1150 TCTCAA TCT ATA AAT GAT GAT ATA GTT AGA AAA GAA GAT TAT ATA CAT 3504 Ser GlnSer Ile Asn Asp Asp Ile Val Arg Lys Glu Asp Tyr Ile His 1155 1160 1165CTA GAT TTG GTA CTT CAC CAT GAA GAG TGG AGA GTA TAT GCC TAT AAA 3552 LeuAsp Leu Val Leu His His Glu Glu Trp Arg Val Tyr Ala Tyr Lys 1170 11751180 TAT TTT AAG GAA CAG GAA GAA AAA TTG TTT TTA TCT ATT ATA AGT GAT3600 Tyr Phe Lys Glu Gln Glu Glu Lys Leu Phe Leu Ser Ile Ile Ser Asp1185 1190 1195 1200 TCT AAT GAA TTT TAT AAG ACT ATA GAA ATA AAA GAA TATGAT GAA CAG 3648 Ser Asn Glu Phe Tyr Lys Thr Ile Glu Ile Lys Glu Tyr AspGlu Gln 1205 1210 1215 CCA TCA TAT AGT TGT CAG TTG CTT TTT AAA AAA GATGAA GAA AGT ACT 3696 Pro Ser Tyr Ser Cys Gln Leu Leu Phe Lys Lys Asp GluGlu Ser Thr 1220 1225 1230 GAT GAT ATA GGA TTG ATT GGT ATT CAT CGT TTCTAC GAA TCT GGA GTT 3744 Asp Asp Ile Gly Leu Ile Gly Ile His Arg Phe TyrGlu Ser Gly Val 1235 1240 1245 TTA CGT AAA AAG TAT AAA GAT TAT TTT TGTATA AGT AAA TGG TAC TTA 3792 Leu Arg Lys Lys Tyr Lys Asp Tyr Phe Cys IleSer Lys Trp Tyr Leu 1250 1255 1260 AAA GAG GTA AAA AGG AAA CCA TAT AAGTCA AAT TTG GGA TGT AAT TGG 3840 Lys Glu Val Lys Arg Lys Pro Tyr Lys SerAsn Leu Gly Cys Asn Trp 1265 1270 1275 1280 CAG TTT ATT CCT AAA GAT GAAGGG TGG ACT GAA TAA 3876 Gln Phe Ile Pro Lys Asp Glu Gly Trp Thr Glu1285 1290 1291 amino acids amino acid linear protein 40 Met Pro Val ThrIle Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn 1 5 10 15 Asp Asn IleIle Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg 20 25 30 Tyr Tyr LysAla Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu 35 40 45 Arg Tyr ThrPhe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly 50 55 60 Ile Phe AsnArg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn 65 70 75 80 Thr AsnAsp Lys Lys Asn Ile Phe Phe Gln Thr Leu Ile Lys Leu Phe 85 90 95 Asn ArgIle Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile 100 105 110 IleAsn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu 115 120 125Phe Asn Thr Asn Ile Ala Ser Val Thr Val Asn Lys Leu Ile Ser Asn 130 135140 Pro Gly Glu Val Glu Arg Lys Lys Gly Ile Phe Ala Asn Leu Ile Ile 145150 155 160 Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu Thr Ile Asp IleGly 165 170 175 Ile Gln Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly IleMet Gln 180 185 190 Met Lys Phe Cys Pro Glu Tyr Val Ser Val Phe Asn AsnVal Gln Glu 195 200 205 Asn Lys Gly Ala Ser Ile Phe Asn Arg Arg Gly TyrPhe Ser Asp Pro 210 215 220 Ala Leu Ile Leu Met His Glu Leu Ile His ValLeu His Gly Leu Tyr 225 230 235 240 Gly Ile Lys Val Asp Asp Leu Pro IleVal Pro Asn Glu Lys Lys Phe 245 250 255 Phe Met Gln Ser Thr Asp Thr IleGln Ala Glu Glu Leu Tyr Thr Phe 260 265 270 Gly Gly Gln Asp Pro Ser IleIle Ser Pro Ser Thr Asp Lys Ser Ile 275 280 285 Tyr Asp Lys Val Leu GlnAsn Phe Arg Gly Ile Val Asp Arg Leu Asn 290 295 300 Lys Val Leu Val CysIle Ser Asp Pro Asn Ile Asn Ile Asn Ile Tyr 305 310 315 320 Lys Asn LysPhe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly 325 330 335 Lys TyrSer Ile Asp Val Glu Ser Phe Asn Lys Leu Tyr Lys Ser Leu 340 345 350 MetLeu Gly Phe Thr Glu Ile Asn Ile Ala Glu Asn Tyr Lys Ile Lys 355 360 365Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys Ile Lys 370 375380 Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu Gly Phe Asn Ile 385390 395 400 Ser Asp Lys Asn Met Gly Lys Glu Tyr Arg Gly Gln Asn Lys AlaIle 405 410 415 Asn Lys Gln Ala Tyr Glu Glu Ile Ser Lys Glu His Leu AlaVal Tyr 420 425 430 Lys Ile Gln Met Cys Lys Ser Val Lys Val Pro Gly IleCys Ile Asp 435 440 445 Val Asp Asn Glu Asn Leu Phe Phe Ile Ala Asp LysAsn Ser Phe Ser 450 455 460 Asp Asp Leu Ser Lys Asn Glu Arg Val Glu TyrAsn Thr Gln Asn Asn 465 470 475 480 Tyr Ile Gly Asn Asp Phe Pro Ile AsnGlu Leu Ile Leu Asp Thr Asp 485 490 495 Leu Ile Ser Lys Ile Glu Leu ProSer Glu Asn Thr Glu Ser Leu Thr 500 505 510 Asp Phe Asn Val Asp Val ProVal Tyr Glu Lys Gln Pro Ala Ile Lys 515 520 525 Lys Val Phe Thr Asp GluAsn Thr Ile Phe Gln Tyr Leu Tyr Ser Gln 530 535 540 Thr Phe Pro Leu AsnIle Arg Asp Ile Ser Leu Thr Ser Ser Phe Asp 545 550 555 560 Asp Ala LeuLeu Val Ser Ser Lys Val Tyr Ser Phe Phe Ser Met Asp 565 570 575 Tyr IleLys Thr Ala Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly 580 585 590 TrpVal Lys Gln Ile Val Asp Asp Phe Val Ile Glu Ala Asn Lys Ser 595 600 605Ser Thr Met Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Ile 610 615620 Gly Leu Ala Leu Asn Val Gly Asp Glu Thr Ala Lys Gly Asn Phe Glu 625630 635 640 Ser Ala Phe Glu Ile Ala Gly Ser Ser Ile Leu Leu Glu Phe IlePro 645 650 655 Glu Leu Leu Ile Pro Val Val Gly Val Phe Leu Leu Glu SerTyr Ile 660 665 670 Asp Asn Lys Asn Lys Ile Ile Lys Thr Ile Asp Asn AlaLeu Thr Lys 675 680 685 Arg Val Glu Lys Trp Ile Asp Met Tyr Gly Leu IleVal Ala Gln Trp 690 695 700 Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr IleLys Glu Gly Met Tyr 705 710 715 720 Lys Ala Leu Asn Tyr Gln Ala Gln AlaLeu Glu Glu Ile Ile Lys Tyr 725 730 735 Lys Tyr Asn Ile Tyr Ser Glu GluGlu Lys Ser Asn Ile Asn Ile Asn 740 745 750 Phe Asn Asp Ile Asn Ser LysLeu Asn Asp Gly Ile Asn Gln Ala Met 755 760 765 Asp Asn Ile Asn Asp PheIle Asn Glu Cys Ser Val Ser Tyr Leu Met 770 775 780 Lys Lys Met Ile ProLeu Ala Val Lys Lys Leu Leu Asp Phe Asp Asn 785 790 795 800 Thr Leu LysLys Asn Leu Leu Asn Tyr Ile Asp Glu Asn Lys Leu Tyr 805 810 815 Leu IleGly Ser Val Glu Asp Glu Lys Ser Lys Val Asp Lys Tyr Leu 820 825 830 LysThr Ile Ile Pro Phe Asp Leu Ser Thr Tyr Ser Asn Ile Glu Ile 835 840 845Leu Ile Lys Ile Phe Asn Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile 850 855860 Ile Leu Asn Leu Arg Tyr Arg Asp Asn Asn Leu Ile Asp Leu Ser Gly 865870 875 880 Tyr Gly Ala Lys Val Glu Val Tyr Asp Gly Val Lys Leu Asn AspLys 885 890 895 Asn Gln Phe Lys Leu Thr Ser Ser Ala Asp Ser Lys Ile ArgVal Thr 900 905 910 Gln Asn Gln Asn Ile Ile Phe Asn Ser Met Phe Leu AspPhe Ser Val 915 920 925 Ser Phe Trp Ile Arg Ile Pro Lys Tyr Arg Asn AspAsp Ile Gln Asn 930 935 940 Tyr Ile His Asn Glu Tyr Thr Ile Ile Asn CysMet Lys Asn Asn Ser 945 950 955 960 Gly Trp Lys Ile Ser Ile Arg Gly AsnArg Ile Ile Trp Thr Leu Ile 965 970 975 Asp Ile Asn Gly Lys Thr Lys SerVal Phe Phe Glu Tyr Asn Ile Arg 980 985 990 Glu Asp Ile Ser Glu Tyr IleAsn Arg Trp Phe Phe Val Thr Ile Thr 995 1000 1005 Asn Asn Leu Asp AsnAla Lys Ile Tyr Ile Asn Gly Thr Leu Glu Ser 1010 1015 1020 Asn Met AspIle Lys Asp Ile Gly Glu Val Ile Val Asn Gly Glu Ile 1025 1030 1035 1040Thr Phe Lys Leu Asp Gly Asp Val Asp Arg Thr Gln Phe Ile Trp Met 10451050 1055 Lys Tyr Phe Ser Ile Phe Asn Thr Gln Leu Asn Gln Ser Asn IleLys 1060 1065 1070 Glu Ile Tyr Lys Ile Gln Ser Tyr Ser Glu Tyr Leu LysAsp Phe Trp 1075 1080 1085 Gly Asn Pro Leu Met Tyr Asn Lys Glu Tyr TyrMet Phe Asn Ala Gly 1090 1095 1100 Asn Lys Asn Ser Tyr Ile Lys Leu ValLys Asp Ser Ser Val Gly Glu 1105 1110 1115 1120 Ile Leu Ile Arg Ser LysTyr Asn Gln Asn Ser Asn Tyr Ile Asn Tyr 1125 1130 1135 Arg Asn Leu TyrIle Gly Glu Lys Phe Ile Ile Arg Arg Glu Ser Asn 1140 1145 1150 Ser GlnSer Ile Asn Asp Asp Ile Val Arg Lys Glu Asp Tyr Ile His 1155 1160 1165Leu Asp Leu Val Leu His His Glu Glu Trp Arg Val Tyr Ala Tyr Lys 11701175 1180 Tyr Phe Lys Glu Gln Glu Glu Lys Leu Phe Leu Ser Ile Ile SerAsp 1185 1190 1195 1200 Ser Asn Glu Phe Tyr Lys Thr Ile Glu Ile Lys GluTyr Asp Glu Gln 1205 1210 1215 Pro Ser Tyr Ser Cys Gln Leu Leu Phe LysLys Asp Glu Glu Ser Thr 1220 1225 1230 Asp Asp Ile Gly Leu Ile Gly IleHis Arg Phe Tyr Glu Ser Gly Val 1235 1240 1245 Leu Arg Lys Lys Tyr LysAsp Tyr Phe Cys Ile Ser Lys Trp Tyr Leu 1250 1255 1260 Lys Glu Val LysArg Lys Pro Tyr Lys Ser Asn Leu Gly Cys Asn Trp 1265 1270 1275 1280 GlnPhe Ile Pro Lys Asp Glu Gly Trp Thr Glu 1285 1290 3876 base pairsnucleic acid double linear DNA (genomic) CDS 1..3873 41 ATG CCA GTT ACAATA AAT AAT TTT AAT TAT AAT GAT CCT ATT GAT AAT 48 Met Pro Val Thr IleAsn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn 1 5 10 15 AAT AAT ATT ATTATG ATG GAG CCT CCA TTT GCG AGA GGT ACG GGG AGA 96 Asn Asn Ile Ile MetMet Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg 20 25 30 TAT TAT AAA GCT TTTAAA ATC ACA GAT CGT ATT TGG ATA ATA CCG GAA 144 Tyr Tyr Lys Ala Phe LysIle Thr Asp Arg Ile Trp Ile Ile Pro Glu 35 40 45 AGA TAT ACT TTT GGA TATAAA CCT GAG GAT TTT AAT AAA AGT TCC GGT 192 Arg Tyr Thr Phe Gly Tyr LysPro Glu Asp Phe Asn Lys Ser Ser Gly 50 55 60 ATT TTT AAT AGA GAT GTT TGTGAA TAT TAT GAT CCA GAT TAC TTA AAT 240 Ile Phe Asn Arg Asp Val Cys GluTyr Tyr Asp Pro Asp Tyr Leu Asn 65 70 75 80 ACT AAT GAT AAA AAG AAT ATATTT TTA CAA ACA ATG ATC AAG TTA TTT 288 Thr Asn Asp Lys Lys Asn Ile PheLeu Gln Thr Met Ile Lys Leu Phe 85 90 95 AAT AGA ATC AAA TCA AAA CCA TTGGGT GAA AAG TTA TTA GAG ATG ATT 336 Asn Arg Ile Lys Ser Lys Pro Leu GlyGlu Lys Leu Leu Glu Met Ile 100 105 110 ATA AAT GGT ATA CCT TAT CTT GGAGAT AGA CGT GTT CCA CTC GAA GAG 384 Ile Asn Gly Ile Pro Tyr Leu Gly AspArg Arg Val Pro Leu Glu Glu 115 120 125 TTT AAC ACA AAC ATT GCT AGT GTAACT GTT AAT AAA TTA ATC AGT AAT 432 Phe Asn Thr Asn Ile Ala Ser Val ThrVal Asn Lys Leu Ile Ser Asn 130 135 140 CCA GGA GAA GTG GAG CGA AAA AAAGGT ATT TTC GCA AAT TTA ATA ATA 480 Pro Gly Glu Val Glu Arg Lys Lys GlyIle Phe Ala Asn Leu Ile Ile 145 150 155 160 TTT GGA CCT GGG CCA GTT TTAAAT GAA AAT GAG ACT ATA GAT ATA GGT 528 Phe Gly Pro Gly Pro Val Leu AsnGlu Asn Glu Thr Ile Asp Ile Gly 165 170 175 ATA CAA AAT CAT TTT GCA TCAAGG GAA GGC TTC GGG GGT ATA ATG CAA 576 Ile Gln Asn His Phe Ala Ser ArgGlu Gly Phe Gly Gly Ile Met Gln 180 185 190 ATG AAG TTT TGC CCA GAA TATGTA AGC GTA TTT AAT AAT GTT CAA GAA 624 Met Lys Phe Cys Pro Glu Tyr ValSer Val Phe Asn Asn Val Gln Glu 195 200 205 AAC AAA GGC GCA AGT ATA TTTAAT AGA CGT GGA TAT TTT TCA GAT CCA 672 Asn Lys Gly Ala Ser Ile Phe AsnArg Arg Gly Tyr Phe Ser Asp Pro 210 215 220 GCC TTG ATA TTA ATG CAT GAACTT ATA CAT GTT TTA CAT GGA TTA TAT 720 Ala Leu Ile Leu Met His Glu LeuIle His Val Leu His Gly Leu Tyr 225 230 235 240 GGC ATT AAA GTA GAT GATTTA CCA ATT GTA CCA AAT GAA AAA AAA TTT 768 Gly Ile Lys Val Asp Asp LeuPro Ile Val Pro Asn Glu Lys Lys Phe 245 250 255 TTT ATG CAA TCT ACA GATGCT ATA CAG GCA GAA GAA CTA TAT ACA TTT 816 Phe Met Gln Ser Thr Asp AlaIle Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270 GGA GGA CAA GAT CCC AGCATC ATA ACT CCT TCT ACG GAT AAA AGT ATC 864 Gly Gly Gln Asp Pro Ser IleIle Thr Pro Ser Thr Asp Lys Ser Ile 275 280 285 TAT GAT AAA GTT TTG CAAAAT TTT AGA GGG ATA GTT GAT AGA CTT AAC 912 Tyr Asp Lys Val Leu Gln AsnPhe Arg Gly Ile Val Asp Arg Leu Asn 290 295 300 AAG GTT TTA GTT TGC ATATCA GAT CCT AAC ATT AAT ATT AAT ATA TAT 960 Lys Val Leu Val Cys Ile SerAsp Pro Asn Ile Asn Ile Asn Ile Tyr 305 310 315 320 AAA AAT AAA TTT AAAGAT AAA TAT AAA TTC GTT GAA GAT TCT GAG GGA 1008 Lys Asn Lys Phe Lys AspLys Tyr Lys Phe Val Glu Asp Ser Glu Gly 325 330 335 AAA TAT AGT ATA GATGTA GAA AGT TTT GAT AAA TTA TAT AAA AGC TTA 1056 Lys Tyr Ser Ile Asp ValGlu Ser Phe Asp Lys Leu Tyr Lys Ser Leu 340 345 350 ATG TTT GGT TTT ACAGAA ACT AAT ATA GCA GAA AAT TAT AAA ATA AAA 1104 Met Phe Gly Phe Thr GluThr Asn Ile Ala Glu Asn Tyr Lys Ile Lys 355 360 365 ACT AGA GCT TCT TATTTT AGT GAT TCC TTA CCA CCA GTA AAA ATA AAA 1152 Thr Arg Ala Ser Tyr PheSer Asp Ser Leu Pro Pro Val Lys Ile Lys 370 375 380 AAT TTA TTA GAT AATGAA ATC TAT ACT ATA GAG GAA GGG TTT AAT ATA 1200 Asn Leu Leu Asp Asn GluIle Tyr Thr Ile Glu Glu Gly Phe Asn Ile 385 390 395 400 TCT GAT AAA GATATG GAA AAA GAA TAT AGA GGT CAG AAT AAA GCT ATA 1248 Ser Asp Lys Asp MetGlu Lys Glu Tyr Arg Gly Gln Asn Lys Ala Ile 405 410 415 AAT AAA CAA GCTTAT GAA GAA ATT AGC AAG GAG CAT TTG GCT GTA TAT 1296 Asn Lys Gln Ala TyrGlu Glu Ile Ser Lys Glu His Leu Ala Val Tyr 420 425 430 AAG ATA CAA ATGTGT AAA AGT GTT AAA GCT CCA GGA ATA TGT ATT GAT 1344 Lys Ile Gln Met CysLys Ser Val Lys Ala Pro Gly Ile Cys Ile Asp 435 440 445 GTT GAT AAT GAAGAT TTG TTC TTT ATA GCT GAT AAA AAT AGT TTT TCA 1392 Val Asp Asn Glu AspLeu Phe Phe Ile Ala Asp Lys Asn Ser Phe Ser 450 455 460 GAT GAT TTA TCTAAA AAC GAA AGA ATA GAA TAT AAT ACA CAG AGT AAT 1440 Asp Asp Leu Ser LysAsn Glu Arg Ile Glu Tyr Asn Thr Gln Ser Asn 465 470 475 480 TAT ATA GAAAAT GAC TTC CCT ATA AAT GAA TTA ATT TTA GAT ACT GAT 1488 Tyr Ile Glu AsnAsp Phe Pro Ile Asn Glu Leu Ile Leu Asp Thr Asp 485 490 495 TTA ATA AGTAAA ATA GAA TTA CCA AGT GAA AAT ACA GAA TCA CTT ACT 1536 Leu Ile Ser LysIle Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr 500 505 510 GAT TTT AATGTA GAT GTT CCA GTA TAT GAA AAA CAA CCC GCT ATA AAA 1584 Asp Phe Asn ValAsp Val Pro Val Tyr Glu Lys Gln Pro Ala Ile Lys 515 520 525 AAA ATT TTTACA GAT GAA AAT ACC ATC TTT CAA TAT TTA TAC TCT CAG 1632 Lys Ile Phe ThrAsp Glu Asn Thr Ile Phe Gln Tyr Leu Tyr Ser Gln 530 535 540 ACA TTT CTCTTA GAT ATA AGA GAT ATA AGT TTA ACA TCT TCA TTT GAT 1680 Thr Phe Leu LeuAsp Ile Arg Asp Ile Ser Leu Thr Ser Ser Phe Asp 545 550 555 560 GAT GCATTA TTA TTT TCT AAC AAA GTT TAT TCA TTT TTT TCT ATG GAT 1728 Asp Ala LeuLeu Phe Ser Asn Lys Val Tyr Ser Phe Phe Ser Met Asp 565 570 575 TAT ATTAAA ACT GCT AAT AAA GTG GTA GAA GCA GGA TTA TTT GCA GGT 1776 Tyr Ile LysThr Ala Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly 580 585 590 TGG GTGAAA CAG ATA GTA AAT GAT TTT GTA ATC GAA GCT AAT AAA AGC 1824 Trp Val LysGln Ile Val Asn Asp Phe Val Ile Glu Ala Asn Lys Ser 595 600 605 AAT ACTATG GAT AAA ATT GCA GAT ATA TCT CTA ATT GTT CCT TAT ATA 1872 Asn Thr MetAsp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Ile 610 615 620 GGA TTAGCT TTA AAT GTA GGA AAT GAA ACA GCT AAA GGA AAT TTT GAA 1920 Gly Leu AlaLeu Asn Val Gly Asn Glu Thr Ala Lys Gly Asn Phe Glu 625 630 635 640 AATGCT TTT GAG ATT GCA GGA GCC AGT ATT CTA CTA GAA TTT ATA CCA 1968 Asn AlaPhe Glu Ile Ala Gly Ala Ser Ile Leu Leu Glu Phe Ile Pro 645 650 655 GAACTT TTA ATA CCT GTA GTT GGA GCC TTT TTA TTA GAA TCA TAT ATT 2016 Glu LeuLeu Ile Pro Val Val Gly Ala Phe Leu Leu Glu Ser Tyr Ile 660 665 670 GACAAT AAA AAT AAA ATT ATT AAA ACA ATA GAT AAT GCT TTA ACT AAA 2064 Asp AsnLys Asn Lys Ile Ile Lys Thr Ile Asp Asn Ala Leu Thr Lys 675 680 685 AGAAAT GAA AAA TGG AGT GAT ATG TAC GGA TTA ATA GTA GCG CAA TGG 2112 Arg AsnGlu Lys Trp Ser Asp Met Tyr Gly Leu Ile Val Ala Gln Trp 690 695 700 CTCTCA ACA GTT AAT ACT CAA TTT TAT ACA ATA AAA GAG GGA ATG TAT 2160 Leu SerThr Val Asn Thr Gln Phe Tyr Thr Ile Lys Glu Gly Met Tyr 705 710 715 720AAG GCT TTA AAT TAT CAA GCA CAA GCA TTG GAA GAA ATA ATA AAA TAC 2208 LysAla Leu Asn Tyr Gln Ala Gln Ala Leu Glu Glu Ile Ile Lys Tyr 725 730 735AGA TAT AAT ATA TAT TCT GAA AAA GAA AAG TCA AAT ATT AAC ATC GAT 2256 ArgTyr Asn Ile Tyr Ser Glu Lys Glu Lys Ser Asn Ile Asn Ile Asp 740 745 750TTT AAT GAT ATA AAT TCT AAA CTT AAT GAG GGT ATT AAC CAA GCT ATA 2304 PheAsn Asp Ile Asn Ser Lys Leu Asn Glu Gly Ile Asn Gln Ala Ile 755 760 765GAT AAT ATA AAT AAT TTT ATA AAT GGA TGT TCT GTA TCA TAT TTA ATG 2352 AspAsn Ile Asn Asn Phe Ile Asn Gly Cys Ser Val Ser Tyr Leu Met 770 775 780AAA AAA ATG ATT CCA TTA GCT GTA GAA AAA TTA CTA GAC TTT GAT AAT 2400 LysLys Met Ile Pro Leu Ala Val Glu Lys Leu Leu Asp Phe Asp Asn 785 790 795800 ACT CTC AAA AAA AAT TTG TTA AAT TAT ATA GAT GAA AAT AAA TTA TAT 2448Thr Leu Lys Lys Asn Leu Leu Asn Tyr Ile Asp Glu Asn Lys Leu Tyr 805 810815 TTG ATT GGA AGT GCA GAA TAT GAA AAA TCA AAA GTA AAT AAA TAC TTG 2496Leu Ile Gly Ser Ala Glu Tyr Glu Lys Ser Lys Val Asn Lys Tyr Leu 820 825830 AAA ACC ATT ATG CCG TTT GAT CTT TCA ATA TAT ACC AAT GAT ACA ATA 2544Lys Thr Ile Met Pro Phe Asp Leu Ser Ile Tyr Thr Asn Asp Thr Ile 835 840845 CTA ATA GAA ATG TTT AAT AAA TAT AAT AGC GAA ATT TTA AAT AAT ATT 2592Leu Ile Glu Met Phe Asn Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile 850 855860 ATC TTA AAT TTA AGA TAT AAG GAT AAT AAT TTA ATA GAT TTA TCA GGA 2640Ile Leu Asn Leu Arg Tyr Lys Asp Asn Asn Leu Ile Asp Leu Ser Gly 865 870875 880 TAT GGG GCA AAG GTA GAG GTA TAT GAT GGA GTC GAG CTT AAT GAT AAA2688 Tyr Gly Ala Lys Val Glu Val Tyr Asp Gly Val Glu Leu Asn Asp Lys 885890 895 AAT CAA TTT AAA TTA ACT AGT TCA GCA AAT AGT AAG ATT AGA GTG ACT2736 Asn Gln Phe Lys Leu Thr Ser Ser Ala Asn Ser Lys Ile Arg Val Thr 900905 910 CAA AAT CAG AAT ATC ATA TTT AAT AGT GTG TTC CTT GAT TTT AGC GTT2784 Gln Asn Gln Asn Ile Ile Phe Asn Ser Val Phe Leu Asp Phe Ser Val 915920 925 AGC TTT TGG ATA AGA ATA CCT AAA TAT AAG AAT GAT GGT ATA CAA AAT2832 Ser Phe Trp Ile Arg Ile Pro Lys Tyr Lys Asn Asp Gly Ile Gln Asn 930935 940 TAT ATT CAT AAT GAA TAT ACA ATA ATT AAT TGT ATG AAA AAT AAT TCG2880 Tyr Ile His Asn Glu Tyr Thr Ile Ile Asn Cys Met Lys Asn Asn Ser 945950 955 960 GGC TGG AAA ATA TCT ATT AGG GGT AAT AGG ATA ATA TGG ACT TTAATT 2928 Gly Trp Lys Ile Ser Ile Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile965 970 975 GAT ATA AAT GGA AAA ACC AAA TCG GTA TTT TTT GAA TAT AAC ATAAGA 2976 Asp Ile Asn Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn Ile Arg980 985 990 GAA GAT ATA TCA GAG TAT ATA AAT AGA TGG TTT TTT GTA ACT ATTACT 3024 Glu Asp Ile Ser Glu Tyr Ile Asn Arg Trp Phe Phe Val Thr Ile Thr995 1000 1005 AAT AAT TTG AAT AAC GCT AAA ATT TAT ATT AAT GGT AAG CTAGAA TCA 3072 Asn Asn Leu Asn Asn Ala Lys Ile Tyr Ile Asn Gly Lys Leu GluSer 1010 1015 1020 AAT ACA GAT ATT AAA GAT ATA AGA GAA GTT ATT GCT AATGGT GAA ATA 3120 Asn Thr Asp Ile Lys Asp Ile Arg Glu Val Ile Ala Asn GlyGlu Ile 1025 1030 1035 1040 ATA TTT AAA TTA GAT GGT GAT ATA GAT AGA ACACAA TTT ATT TGG ATG 3168 Ile Phe Lys Leu Asp Gly Asp Ile Asp Arg Thr GlnPhe Ile Trp Met 1045 1050 1055 AAA TAT TTC AGT ATT TTT AAT ACG GAA TTAAGT CAA TCA AAT ATT GAA 3216 Lys Tyr Phe Ser Ile Phe Asn Thr Glu Leu SerGln Ser Asn Ile Glu 1060 1065 1070 GAA AGA TAT AAA ATT CAA TCA TAT AGCGAA TAT TTA AAA GAT TTT TGG 3264 Glu Arg Tyr Lys Ile Gln Ser Tyr Ser GluTyr Leu Lys Asp Phe Trp 1075 1080 1085 GGA AAT CCT TTA ATG TAC AAT AAAGAA TAT TAT ATG TTT AAT GCG GGG 3312 Gly Asn Pro Leu Met Tyr Asn Lys GluTyr Tyr Met Phe Asn Ala Gly 1090 1095 1100 AAT AAA AAT TCA TAT ATT AAACTA AAG AAA GAT TCA CCT GTA GGT GAA 3360 Asn Lys Asn Ser Tyr Ile Lys LeuLys Lys Asp Ser Pro Val Gly Glu 1105 1110 1115 1120 ATT TTA ACA CGT AGCAAA TAT AAT CAA AAT TCT AAA TAT ATA AAT TAT 3408 Ile Leu Thr Arg Ser LysTyr Asn Gln Asn Ser Lys Tyr Ile Asn Tyr 1125 1130 1135 AGA GAT TTA TATATT GGA GAA AAA TTT ATT ATA AGA AGA AAG TCA AAT 3456 Arg Asp Leu Tyr IleGly Glu Lys Phe Ile Ile Arg Arg Lys Ser Asn 1140 1145 1150 TCT CAA TCTATA AAT GAT GAT ATA GTT AGA AAA GAA GAT TAT ATA TAT 3504 Ser Gln Ser IleAsn Asp Asp Ile Val Arg Lys Glu Asp Tyr Ile Tyr 1155 1160 1165 CTA GATTTT TTT AAT TTA AAT CAA GAG TGG AGA GTA TAT ACC TAT AAA 3552 Leu Asp PhePhe Asn Leu Asn Gln Glu Trp Arg Val Tyr Thr Tyr Lys 1170 1175 1180 TATTTT AAG AAA GAG GAA GAA AAA TTG TTT TTA GCT CCT ATA AGT GAT 3600 Tyr PheLys Lys Glu Glu Glu Lys Leu Phe Leu Ala Pro Ile Ser Asp 1185 1190 11951200 TCT GAT GAG TTT TAC AAT ACT ATA CAA ATA AAA GAA TAT GAT GAA CAG3648 Ser Asp Glu Phe Tyr Asn Thr Ile Gln Ile Lys Glu Tyr Asp Glu Gln1205 1210 1215 CCA ACA TAT AGT TGT CAG TTG CTT TTT AAA AAA GAT GAA GAAAGT ACT 3696 Pro Thr Tyr Ser Cys Gln Leu Leu Phe Lys Lys Asp Glu Glu SerThr 1220 1225 1230 GAT GAG ATA GGA TTG ATT GGT ATT CAT CGT TTC TAC GAATCT GGA ATT 3744 Asp Glu Ile Gly Leu Ile Gly Ile His Arg Phe Tyr Glu SerGly Ile 1235 1240 1245 GTA TTT GAA GAG TAT AAA GAT TAT TTT TGT ATA AGTAAA TGG TAC TTA 3792 Val Phe Glu Glu Tyr Lys Asp Tyr Phe Cys Ile Ser LysTrp Tyr Leu 1250 1255 1260 AAA GAG GTA AAA AGG AAA CCA TAT AAT TTA AAATTG GGA TGT AAT TGG 3840 Lys Glu Val Lys Arg Lys Pro Tyr Asn Leu Lys LeuGly Cys Asn Trp 1265 1270 1275 1280 CAG TTT ATT CCT AAA GAT GAA GGG TGGACT GAA TAA 3876 Gln Phe Ile Pro Lys Asp Glu Gly Trp Thr Glu 1285 12901291 amino acids amino acid linear protein 42 Met Pro Val Thr Ile AsnAsn Phe Asn Tyr Asn Asp Pro Ile Asp Asn 1 5 10 15 Asn Asn Ile Ile MetMet Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg 20 25 30 Tyr Tyr Lys Ala PheLys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu 35 40 45 Arg Tyr Thr Phe GlyTyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly 50 55 60 Ile Phe Asn Arg AspVal Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn 65 70 75 80 Thr Asn Asp LysLys Asn Ile Phe Leu Gln Thr Met Ile Lys Leu Phe 85 90 95 Asn Arg Ile LysSer Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile 100 105 110 Ile Asn GlyIle Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu 115 120 125 Phe AsnThr Asn Ile Ala Ser Val Thr Val Asn Lys Leu Ile Ser Asn 130 135 140 ProGly Glu Val Glu Arg Lys Lys Gly Ile Phe Ala Asn Leu Ile Ile 145 150 155160 Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu Thr Ile Asp Ile Gly 165170 175 Ile Gln Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly Ile Met Gln180 185 190 Met Lys Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val GlnGlu 195 200 205 Asn Lys Gly Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe SerAsp Pro 210 215 220 Ala Leu Ile Leu Met His Glu Leu Ile His Val Leu HisGly Leu Tyr 225 230 235 240 Gly Ile Lys Val Asp Asp Leu Pro Ile Val ProAsn Glu Lys Lys Phe 245 250 255 Phe Met Gln Ser Thr Asp Ala Ile Gln AlaGlu Glu Leu Tyr Thr Phe 260 265 270 Gly Gly Gln Asp Pro Ser Ile Ile ThrPro Ser Thr Asp Lys Ser Ile 275 280 285 Tyr Asp Lys Val Leu Gln Asn PheArg Gly Ile Val Asp Arg Leu Asn 290 295 300 Lys Val Leu Val Cys Ile SerAsp Pro Asn Ile Asn Ile Asn Ile Tyr 305 310 315 320 Lys Asn Lys Phe LysAsp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly 325 330 335 Lys Tyr Ser IleAsp Val Glu Ser Phe Asp Lys Leu Tyr Lys Ser Leu 340 345 350 Met Phe GlyPhe Thr Glu Thr Asn Ile Ala Glu Asn Tyr Lys Ile Lys 355 360 365 Thr ArgAla Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys Ile Lys 370 375 380 AsnLeu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu Gly Phe Asn Ile 385 390 395400 Ser Asp Lys Asp Met Glu Lys Glu Tyr Arg Gly Gln Asn Lys Ala Ile 405410 415 Asn Lys Gln Ala Tyr Glu Glu Ile Ser Lys Glu His Leu Ala Val Tyr420 425 430 Lys Ile Gln Met Cys Lys Ser Val Lys Ala Pro Gly Ile Cys IleAsp 435 440 445 Val Asp Asn Glu Asp Leu Phe Phe Ile Ala Asp Lys Asn SerPhe Ser 450 455 460 Asp Asp Leu Ser Lys Asn Glu Arg Ile Glu Tyr Asn ThrGln Ser Asn 465 470 475 480 Tyr Ile Glu Asn Asp Phe Pro Ile Asn Glu LeuIle Leu Asp Thr Asp 485 490 495 Leu Ile Ser Lys Ile Glu Leu Pro Ser GluAsn Thr Glu Ser Leu Thr 500 505 510 Asp Phe Asn Val Asp Val Pro Val TyrGlu Lys Gln Pro Ala Ile Lys 515 520 525 Lys Ile Phe Thr Asp Glu Asn ThrIle Phe Gln Tyr Leu Tyr Ser Gln 530 535 540 Thr Phe Leu Leu Asp Ile ArgAsp Ile Ser Leu Thr Ser Ser Phe Asp 545 550 555 560 Asp Ala Leu Leu PheSer Asn Lys Val Tyr Ser Phe Phe Ser Met Asp 565 570 575 Tyr Ile Lys ThrAla Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly 580 585 590 Trp Val LysGln Ile Val Asn Asp Phe Val Ile Glu Ala Asn Lys Ser 595 600 605 Asn ThrMet Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Ile 610 615 620 GlyLeu Ala Leu Asn Val Gly Asn Glu Thr Ala Lys Gly Asn Phe Glu 625 630 635640 Asn Ala Phe Glu Ile Ala Gly Ala Ser Ile Leu Leu Glu Phe Ile Pro 645650 655 Glu Leu Leu Ile Pro Val Val Gly Ala Phe Leu Leu Glu Ser Tyr Ile660 665 670 Asp Asn Lys Asn Lys Ile Ile Lys Thr Ile Asp Asn Ala Leu ThrLys 675 680 685 Arg Asn Glu Lys Trp Ser Asp Met Tyr Gly Leu Ile Val AlaGln Trp 690 695 700 Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile Lys GluGly Met Tyr 705 710 715 720 Lys Ala Leu Asn Tyr Gln Ala Gln Ala Leu GluGlu Ile Ile Lys Tyr 725 730 735 Arg Tyr Asn Ile Tyr Ser Glu Lys Glu LysSer Asn Ile Asn Ile Asp 740 745 750 Phe Asn Asp Ile Asn Ser Lys Leu AsnGlu Gly Ile Asn Gln Ala Ile 755 760 765 Asp Asn Ile Asn Asn Phe Ile AsnGly Cys Ser Val Ser Tyr Leu Met 770 775 780 Lys Lys Met Ile Pro Leu AlaVal Glu Lys Leu Leu Asp Phe Asp Asn 785 790 795 800 Thr Leu Lys Lys AsnLeu Leu Asn Tyr Ile Asp Glu Asn Lys Leu Tyr 805 810 815 Leu Ile Gly SerAla Glu Tyr Glu Lys Ser Lys Val Asn Lys Tyr Leu 820 825 830 Lys Thr IleMet Pro Phe Asp Leu Ser Ile Tyr Thr Asn Asp Thr Ile 835 840 845 Leu IleGlu Met Phe Asn Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile 850 855 860 IleLeu Asn Leu Arg Tyr Lys Asp Asn Asn Leu Ile Asp Leu Ser Gly 865 870 875880 Tyr Gly Ala Lys Val Glu Val Tyr Asp Gly Val Glu Leu Asn Asp Lys 885890 895 Asn Gln Phe Lys Leu Thr Ser Ser Ala Asn Ser Lys Ile Arg Val Thr900 905 910 Gln Asn Gln Asn Ile Ile Phe Asn Ser Val Phe Leu Asp Phe SerVal 915 920 925 Ser Phe Trp Ile Arg Ile Pro Lys Tyr Lys Asn Asp Gly IleGln Asn 930 935 940 Tyr Ile His Asn Glu Tyr Thr Ile Ile Asn Cys Met LysAsn Asn Ser 945 950 955 960 Gly Trp Lys Ile Ser Ile Arg Gly Asn Arg IleIle Trp Thr Leu Ile 965 970 975 Asp Ile Asn Gly Lys Thr Lys Ser Val PhePhe Glu Tyr Asn Ile Arg 980 985 990 Glu Asp Ile Ser Glu Tyr Ile Asn ArgTrp Phe Phe Val Thr Ile Thr 995 1000 1005 Asn Asn Leu Asn Asn Ala LysIle Tyr Ile Asn Gly Lys Leu Glu Ser 1010 1015 1020 Asn Thr Asp Ile LysAsp Ile Arg Glu Val Ile Ala Asn Gly Glu Ile 1025 1030 1035 1040 Ile PheLys Leu Asp Gly Asp Ile Asp Arg Thr Gln Phe Ile Trp Met 1045 1050 1055Lys Tyr Phe Ser Ile Phe Asn Thr Glu Leu Ser Gln Ser Asn Ile Glu 10601065 1070 Glu Arg Tyr Lys Ile Gln Ser Tyr Ser Glu Tyr Leu Lys Asp PheTrp 1075 1080 1085 Gly Asn Pro Leu Met Tyr Asn Lys Glu Tyr Tyr Met PheAsn Ala Gly 1090 1095 1100 Asn Lys Asn Ser Tyr Ile Lys Leu Lys Lys AspSer Pro Val Gly Glu 1105 1110 1115 1120 Ile Leu Thr Arg Ser Lys Tyr AsnGln Asn Ser Lys Tyr Ile Asn Tyr 1125 1130 1135 Arg Asp Leu Tyr Ile GlyGlu Lys Phe Ile Ile Arg Arg Lys Ser Asn 1140 1145 1150 Ser Gln Ser IleAsn Asp Asp Ile Val Arg Lys Glu Asp Tyr Ile Tyr 1155 1160 1165 Leu AspPhe Phe Asn Leu Asn Gln Glu Trp Arg Val Tyr Thr Tyr Lys 1170 1175 1180Tyr Phe Lys Lys Glu Glu Glu Lys Leu Phe Leu Ala Pro Ile Ser Asp 11851190 1195 1200 Ser Asp Glu Phe Tyr Asn Thr Ile Gln Ile Lys Glu Tyr AspGlu Gln 1205 1210 1215 Pro Thr Tyr Ser Cys Gln Leu Leu Phe Lys Lys AspGlu Glu Ser Thr 1220 1225 1230 Asp Glu Ile Gly Leu Ile Gly Ile His ArgPhe Tyr Glu Ser Gly Ile 1235 1240 1245 Val Phe Glu Glu Tyr Lys Asp TyrPhe Cys Ile Ser Lys Trp Tyr Leu 1250 1255 1260 Lys Glu Val Lys Arg LysPro Tyr Asn Leu Lys Leu Gly Cys Asn Trp 1265 1270 1275 1280 Gln Phe IlePro Lys Asp Glu Gly Trp Thr Glu 1285 1290 1526 base pairs nucleic aciddouble linear other nucleic acid /desc = “DNA” CDS 108..1523 43AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACC ATG GGC CAT 116 MetGly His 1 CAT CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT ATC GAAGGT 164 His His His His His His His His His Ser Ser Gly His Ile Glu Gly5 10 15 CGT CAT ATG GCT AGC ATG GCT GAT ACA ATA CTA ATA GAA ATG TTT AAT212 Arg His Met Ala Ser Met Ala Asp Thr Ile Leu Ile Glu Met Phe Asn 2025 30 35 AAA TAT AAT AGC GAA ATT TTA AAT AAT ATT ATC TTA AAT TTA AGA TAT260 Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile Ile Leu Asn Leu Arg Tyr 4045 50 AGA GAT AAT AAT TTA ATA GAT TTA TCA GGA TAT GGA GCA AAG GTA GAG308 Arg Asp Asn Asn Leu Ile Asp Leu Ser Gly Tyr Gly Ala Lys Val Glu 5560 65 GTA TAT GAT GGG GTC AAG CTT AAT GAT AAA AAT CAA TTT AAA TTA ACT356 Val Tyr Asp Gly Val Lys Leu Asn Asp Lys Asn Gln Phe Lys Leu Thr 7075 80 AGT TCA GCA GAT AGT AAG ATT AGA GTC ACT CAA AAT CAG AAT ATT ATA404 Ser Ser Ala Asp Ser Lys Ile Arg Val Thr Gln Asn Gln Asn Ile Ile 8590 95 TTT AAT AGT ATG TTC CTT GAT TTT AGC GTT AGC TTT TGG ATA AGG ATA452 Phe Asn Ser Met Phe Leu Asp Phe Ser Val Ser Phe Trp Ile Arg Ile 100105 110 115 CCT AAA TAT AGG AAT GAT GAT ATA CAA AAT TAT ATT CAT AAT GAATAT 500 Pro Lys Tyr Arg Asn Asp Asp Ile Gln Asn Tyr Ile His Asn Glu Tyr120 125 130 ACG ATA ATT AAT TGT ATG AAA AAT AAT TCA GGC TGG AAA ATA TCTATT 548 Thr Ile Ile Asn Cys Met Lys Asn Asn Ser Gly Trp Lys Ile Ser Ile135 140 145 AGG GGT AAT AGG ATA ATA TGG ACC TTA ATT GAT ATA AAT GGA AAAACC 596 Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile Asp Ile Asn Gly Lys Thr150 155 160 AAA TCA GTA TTT TTT GAA TAT AAC ATA AGA GAA GAT ATA TCA GAGTAT 644 Lys Ser Val Phe Phe Glu Tyr Asn Ile Arg Glu Asp Ile Ser Glu Tyr165 170 175 ATA AAT AGA TGG TTT TTT GTA ACT ATT ACT AAT AAT TTG GAT AATGCT 692 Ile Asn Arg Trp Phe Phe Val Thr Ile Thr Asn Asn Leu Asp Asn Ala180 185 190 195 AAA ATT TAT ATT AAT GGC ACG TTA GAA TCA AAT ATG GAT ATTAAA GAT 740 Lys Ile Tyr Ile Asn Gly Thr Leu Glu Ser Asn Met Asp Ile LysAsp 200 205 210 ATA GGA GAA GTT ATT GTT AAT GGT GAA ATA ACA TTT AAA TTAGAT GGT 788 Ile Gly Glu Val Ile Val Asn Gly Glu Ile Thr Phe Lys Leu AspGly 215 220 225 GAT GTA GAT AGA ACA CAA TTT ATT TGG ATG AAA TAT TTT AGTATT TTT 836 Asp Val Asp Arg Thr Gln Phe Ile Trp Met Lys Tyr Phe Ser IlePhe 230 235 240 AAT ACG CAA TTA AAT CAA TCA AAT ATT AAA GAG ATA TAT AAAATT CAA 884 Asn Thr Gln Leu Asn Gln Ser Asn Ile Lys Glu Ile Tyr Lys IleGln 245 250 255 TCA TAT AGC GAA TAC TTA AAA GAT TTT TGG GGA AAT CCT TTAATG TAT 932 Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp Gly Asn Pro Leu MetTyr 260 265 270 275 AAT AAA GAA TAT TAT ATG TTT AAT GCG GGG AAT AAA AATTCA TAT ATT 980 Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly Asn Lys Asn SerTyr Ile 280 285 290 AAA CTA GTG AAA GAT TCA TCT GTA GGT GAA ATA TTA ATACGT AGC AAA 1028 Lys Leu Val Lys Asp Ser Ser Val Gly Glu Ile Leu Ile ArgSer Lys 295 300 305 TAT AAT CAG AAT TCC AAT TAT ATA AAT TAT AGA AAT TTATAT ATT GGA 1076 Tyr Asn Gln Asn Ser Asn Tyr Ile Asn Tyr Arg Asn Leu TyrIle Gly 310 315 320 GAA AAA TTT ATT ATA AGA AGA GAG TCA AAT TCT CAA TCTATA AAT GAT 1124 Glu Lys Phe Ile Ile Arg Arg Glu Ser Asn Ser Gln Ser IleAsn Asp 325 330 335 GAT ATA GTT AGA AAA GAA GAT TAT ATA CAT CTA GAT TTGGTA CTT CAC 1172 Asp Ile Val Arg Lys Glu Asp Tyr Ile His Leu Asp Leu ValLeu His 340 345 350 355 CAT GAA GAG TGG AGA GTA TAT GCC TAT AAA TAT TTTAAG GAA CAG GAA 1220 His Glu Glu Trp Arg Val Tyr Ala Tyr Lys Tyr Phe LysGlu Gln Glu 360 365 370 GAA AAA TTG TTT TTA TCT ATT ATA AGT GAT TCT AATGAA TTT TAT AAG 1268 Glu Lys Leu Phe Leu Ser Ile Ile Ser Asp Ser Asn GluPhe Tyr Lys 375 380 385 ACT ATA GAA ATA AAA GAA TAT GAT GAA CAG CCA TCATAT AGT TGT CAG 1316 Thr Ile Glu Ile Lys Glu Tyr Asp Glu Gln Pro Ser TyrSer Cys Gln 390 395 400 TTG CTT TTT AAA AAA GAT GAA GAA AGT ACT GAT GATATA GGA TTG ATT 1364 Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr Asp Asp IleGly Leu Ile 405 410 415 GGT ATT CAT CGT TTC TAC GAA TCT GGA GTT TTA CGTAAA AAG TAT AAA 1412 Gly Ile His Arg Phe Tyr Glu Ser Gly Val Leu Arg LysLys Tyr Lys 420 425 430 435 GAT TAT TTT TGT ATA AGT AAA TGG TAC TTA AAAGAG GTA AAA AGG AAA 1460 Asp Tyr Phe Cys Ile Ser Lys Trp Tyr Leu Lys GluVal Lys Arg Lys 440 445 450 CCA TAT AAG TCA AAT TTG GGA TGT AAT TGG CAGTTT ATT CCT AAA GAT 1508 Pro Tyr Lys Ser Asn Leu Gly Cys Asn Trp Gln PheIle Pro Lys Asp 455 460 465 GAA GGG TGG ACT GAA TAA 1526 Glu Gly Trp ThrGlu 470 472 amino acids amino acid linear protein 44 Met Gly His His HisHis His His His His His His Ser Ser Gly His 1 5 10 15 Ile Glu Gly ArgHis Met Ala Ser Met Ala Asp Thr Ile Leu Ile Glu 20 25 30 Met Phe Asn LysTyr Asn Ser Glu Ile Leu Asn Asn Ile Ile Leu Asn 35 40 45 Leu Arg Tyr ArgAsp Asn Asn Leu Ile Asp Leu Ser Gly Tyr Gly Ala 50 55 60 Lys Val Glu ValTyr Asp Gly Val Lys Leu Asn Asp Lys Asn Gln Phe 65 70 75 80 Lys Leu ThrSer Ser Ala Asp Ser Lys Ile Arg Val Thr Gln Asn Gln 85 90 95 Asn Ile IlePhe Asn Ser Met Phe Leu Asp Phe Ser Val Ser Phe Trp 100 105 110 Ile ArgIle Pro Lys Tyr Arg Asn Asp Asp Ile Gln Asn Tyr Ile His 115 120 125 AsnGlu Tyr Thr Ile Ile Asn Cys Met Lys Asn Asn Ser Gly Trp Lys 130 135 140Ile Ser Ile Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile Asp Ile Asn 145 150155 160 Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn Ile Arg Glu Asp Ile165 170 175 Ser Glu Tyr Ile Asn Arg Trp Phe Phe Val Thr Ile Thr Asn AsnLeu 180 185 190 Asp Asn Ala Lys Ile Tyr Ile Asn Gly Thr Leu Glu Ser AsnMet Asp 195 200 205 Ile Lys Asp Ile Gly Glu Val Ile Val Asn Gly Glu IleThr Phe Lys 210 215 220 Leu Asp Gly Asp Val Asp Arg Thr Gln Phe Ile TrpMet Lys Tyr Phe 225 230 235 240 Ser Ile Phe Asn Thr Gln Leu Asn Gln SerAsn Ile Lys Glu Ile Tyr 245 250 255 Lys Ile Gln Ser Tyr Ser Glu Tyr LeuLys Asp Phe Trp Gly Asn Pro 260 265 270 Leu Met Tyr Asn Lys Glu Tyr TyrMet Phe Asn Ala Gly Asn Lys Asn 275 280 285 Ser Tyr Ile Lys Leu Val LysAsp Ser Ser Val Gly Glu Ile Leu Ile 290 295 300 Arg Ser Lys Tyr Asn GlnAsn Ser Asn Tyr Ile Asn Tyr Arg Asn Leu 305 310 315 320 Tyr Ile Gly GluLys Phe Ile Ile Arg Arg Glu Ser Asn Ser Gln Ser 325 330 335 Ile Asn AspAsp Ile Val Arg Lys Glu Asp Tyr Ile His Leu Asp Leu 340 345 350 Val LeuHis His Glu Glu Trp Arg Val Tyr Ala Tyr Lys Tyr Phe Lys 355 360 365 GluGln Glu Glu Lys Leu Phe Leu Ser Ile Ile Ser Asp Ser Asn Glu 370 375 380Phe Tyr Lys Thr Ile Glu Ile Lys Glu Tyr Asp Glu Gln Pro Ser Tyr 385 390395 400 Ser Cys Gln Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr Asp Asp Ile405 410 415 Gly Leu Ile Gly Ile His Arg Phe Tyr Glu Ser Gly Val Leu ArgLys 420 425 430 Lys Tyr Lys Asp Tyr Phe Cys Ile Ser Lys Trp Tyr Leu LysGlu Val 435 440 445 Lys Arg Lys Pro Tyr Lys Ser Asn Leu Gly Cys Asn TrpGln Phe Ile 450 455 460 Pro Lys Asp Glu Gly Trp Thr Glu 465 470 1547base pairs nucleic acid double linear DNA (genomic) CDS 108..1523 45AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACC ATG GGC CAT 116 MetGly His 1 CAT CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT ATC GAAGGT 164 His His His His His His His His His Ser Ser Gly His Ile Glu Gly5 10 15 CGT CAT ATG GCT AGC ATG GCT GAT ACA ATA CTA ATA GAA ATG TTT AAT212 Arg His Met Ala Ser Met Ala Asp Thr Ile Leu Ile Glu Met Phe Asn 2025 30 35 AAA TAT AAT AGC GAA ATT TTA AAT AAT ATT ATC TTA AAT TTA AGA TAT260 Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile Ile Leu Asn Leu Arg Tyr 4045 50 AAG GAT AAT AAT TTA ATA GAT TTA TCA GGA TAT GGG GCA AAG GTA GAG308 Lys Asp Asn Asn Leu Ile Asp Leu Ser Gly Tyr Gly Ala Lys Val Glu 5560 65 GTA TAT GAT GGA GTC GAG CTT AAT GAT AAA AAT CAA TTT AAA TTA ACT356 Val Tyr Asp Gly Val Glu Leu Asn Asp Lys Asn Gln Phe Lys Leu Thr 7075 80 AGT TCA GCA AAT AGT AAG ATT AGA GTG ACT CAA AAT CAG AAT ATC ATA404 Ser Ser Ala Asn Ser Lys Ile Arg Val Thr Gln Asn Gln Asn Ile Ile 8590 95 TTT AAT AGT GTG TTC CTT GAT TTT AGC GTT AGC TTT TGG ATA AGA ATA452 Phe Asn Ser Val Phe Leu Asp Phe Ser Val Ser Phe Trp Ile Arg Ile 100105 110 115 CCT AAA TAT AAG AAT GAT GGT ATA CAA AAT TAT ATT CAT AAT GAATAT 500 Pro Lys Tyr Lys Asn Asp Gly Ile Gln Asn Tyr Ile His Asn Glu Tyr120 125 130 ACA ATA ATT AAT TGT ATG AAA AAT AAT TCG GGC TGG AAA ATA TCTATT 548 Thr Ile Ile Asn Cys Met Lys Asn Asn Ser Gly Trp Lys Ile Ser Ile135 140 145 AGG GGT AAT AGG ATA ATA TGG ACT TTA ATT GAT ATA AAT GGA AAAACC 596 Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile Asp Ile Asn Gly Lys Thr150 155 160 AAA TCG GTA TTT TTT GAA TAT AAC ATA AGA GAA GAT ATA TCA GAGTAT 644 Lys Ser Val Phe Phe Glu Tyr Asn Ile Arg Glu Asp Ile Ser Glu Tyr165 170 175 ATA AAT AGA TGG TTT TTT GTA ACT ATT ACT AAT AAT TTG AAT AACGCT 692 Ile Asn Arg Trp Phe Phe Val Thr Ile Thr Asn Asn Leu Asn Asn Ala180 185 190 195 AAA ATT TAT ATT AAT GGT AAG CTA GAA TCA AAT ACA GAT ATTAAA GAT 740 Lys Ile Tyr Ile Asn Gly Lys Leu Glu Ser Asn Thr Asp Ile LysAsp 200 205 210 ATA AGA GAA GTT ATT GCT AAT GGT GAA ATA ATA TTT AAA TTAGAT GGT 788 Ile Arg Glu Val Ile Ala Asn Gly Glu Ile Ile Phe Lys Leu AspGly 215 220 225 GAT ATA GAT AGA ACA CAA TTT ATT TGG ATG AAA TAT TTC AGTATT TTT 836 Asp Ile Asp Arg Thr Gln Phe Ile Trp Met Lys Tyr Phe Ser IlePhe 230 235 240 AAT ACG GAA TTA AGT CAA TCA AAT ATT GAA GAA AGA TAT AAAATT CAA 884 Asn Thr Glu Leu Ser Gln Ser Asn Ile Glu Glu Arg Tyr Lys IleGln 245 250 255 TCA TAT AGC GAA TAT TTA AAA GAT TTT TGG GGA AAT CCT TTAATG TAC 932 Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp Gly Asn Pro Leu MetTyr 260 265 270 275 AAT AAA GAA TAT TAT ATG TTT AAT GCG GGG AAT AAA AATTCA TAT ATT 980 Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly Asn Lys Asn SerTyr Ile 280 285 290 AAA CTA AAG AAA GAT TCA CCT GTA GGT GAA ATT TTA ACACGT AGC AAA 1028 Lys Leu Lys Lys Asp Ser Pro Val Gly Glu Ile Leu Thr ArgSer Lys 295 300 305 TAT AAT CAA AAT TCT AAA TAT ATA AAT TAT AGA GAT TTATAT ATT GGA 1076 Tyr Asn Gln Asn Ser Lys Tyr Ile Asn Tyr Arg Asp Leu TyrIle Gly 310 315 320 GAA AAA TTT ATT ATA AGA AGA AAG TCA AAT TCT CAA TCTATA AAT GAT 1124 Glu Lys Phe Ile Ile Arg Arg Lys Ser Asn Ser Gln Ser IleAsn Asp 325 330 335 GAT ATA GTT AGA AAA GAA GAT TAT ATA TAT CTA GAT TTTTTT AAT TTA 1172 Asp Ile Val Arg Lys Glu Asp Tyr Ile Tyr Leu Asp Phe PheAsn Leu 340 345 350 355 AAT CAA GAG TGG AGA GTA TAT ACC TAT AAA TAT TTTAAG AAA GAG GAA 1220 Asn Gln Glu Trp Arg Val Tyr Thr Tyr Lys Tyr Phe LysLys Glu Glu 360 365 370 GAA AAA TTG TTT TTA GCT CCT ATA AGT GAT TCT GATGAG TTT TAC AAT 1268 Glu Lys Leu Phe Leu Ala Pro Ile Ser Asp Ser Asp GluPhe Tyr Asn 375 380 385 ACT ATA CAA ATA AAA GAA TAT GAT GAA CAG CCA ACATAT AGT TGT CAG 1316 Thr Ile Gln Ile Lys Glu Tyr Asp Glu Gln Pro Thr TyrSer Cys Gln 390 395 400 TTG CTT TTT AAA AAA GAT GAA GAA AGT ACT GAT GAGATA GGA TTG ATT 1364 Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr Asp Glu IleGly Leu Ile 405 410 415 GGT ATT CAT CGT TTC TAC GAA TCT GGA ATT GTA TTTGAA GAG TAT AAA 1412 Gly Ile His Arg Phe Tyr Glu Ser Gly Ile Val Phe GluGlu Tyr Lys 420 425 430 435 GAT TAT TTT TGT ATA AGT AAA TGG TAC TTA AAAGAG GTA AAA AGG AAA 1460 Asp Tyr Phe Cys Ile Ser Lys Trp Tyr Leu Lys GluVal Lys Arg Lys 440 445 450 CCA TAT AAT TTA AAA TTG GGA TGT AAT TGG CAGTTT ATT CCT AAA GAT 1508 Pro Tyr Asn Leu Lys Leu Gly Cys Asn Trp Gln PheIle Pro Lys Asp 455 460 465 GAA GGG TGG ACT GAA TAAAAGCTTG CGGCCGCACTCGAG 1547 Glu Gly Trp Thr Glu 470 472 amino acids amino acid linearprotein 46 Met Gly His His His His His His His His His His Ser Ser GlyHis 1 5 10 15 Ile Glu Gly Arg His Met Ala Ser Met Ala Asp Thr Ile LeuIle Glu 20 25 30 Met Phe Asn Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile IleLeu Asn 35 40 45 Leu Arg Tyr Lys Asp Asn Asn Leu Ile Asp Leu Ser Gly TyrGly Ala 50 55 60 Lys Val Glu Val Tyr Asp Gly Val Glu Leu Asn Asp Lys AsnGln Phe 65 70 75 80 Lys Leu Thr Ser Ser Ala Asn Ser Lys Ile Arg Val ThrGln Asn Gln 85 90 95 Asn Ile Ile Phe Asn Ser Val Phe Leu Asp Phe Ser ValSer Phe Trp 100 105 110 Ile Arg Ile Pro Lys Tyr Lys Asn Asp Gly Ile GlnAsn Tyr Ile His 115 120 125 Asn Glu Tyr Thr Ile Ile Asn Cys Met Lys AsnAsn Ser Gly Trp Lys 130 135 140 Ile Ser Ile Arg Gly Asn Arg Ile Ile TrpThr Leu Ile Asp Ile Asn 145 150 155 160 Gly Lys Thr Lys Ser Val Phe PheGlu Tyr Asn Ile Arg Glu Asp Ile 165 170 175 Ser Glu Tyr Ile Asn Arg TrpPhe Phe Val Thr Ile Thr Asn Asn Leu 180 185 190 Asn Asn Ala Lys Ile TyrIle Asn Gly Lys Leu Glu Ser Asn Thr Asp 195 200 205 Ile Lys Asp Ile ArgGlu Val Ile Ala Asn Gly Glu Ile Ile Phe Lys 210 215 220 Leu Asp Gly AspIle Asp Arg Thr Gln Phe Ile Trp Met Lys Tyr Phe 225 230 235 240 Ser IlePhe Asn Thr Glu Leu Ser Gln Ser Asn Ile Glu Glu Arg Tyr 245 250 255 LysIle Gln Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp Gly Asn Pro 260 265 270Leu Met Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly Asn Lys Asn 275 280285 Ser Tyr Ile Lys Leu Lys Lys Asp Ser Pro Val Gly Glu Ile Leu Thr 290295 300 Arg Ser Lys Tyr Asn Gln Asn Ser Lys Tyr Ile Asn Tyr Arg Asp Leu305 310 315 320 Tyr Ile Gly Glu Lys Phe Ile Ile Arg Arg Lys Ser Asn SerGln Ser 325 330 335 Ile Asn Asp Asp Ile Val Arg Lys Glu Asp Tyr Ile TyrLeu Asp Phe 340 345 350 Phe Asn Leu Asn Gln Glu Trp Arg Val Tyr Thr TyrLys Tyr Phe Lys 355 360 365 Lys Glu Glu Glu Lys Leu Phe Leu Ala Pro IleSer Asp Ser Asp Glu 370 375 380 Phe Tyr Asn Thr Ile Gln Ile Lys Glu TyrAsp Glu Gln Pro Thr Tyr 385 390 395 400 Ser Cys Gln Leu Leu Phe Lys LysAsp Glu Glu Ser Thr Asp Glu Ile 405 410 415 Gly Leu Ile Gly Ile His ArgPhe Tyr Glu Ser Gly Ile Val Phe Glu 420 425 430 Glu Tyr Lys Asp Tyr PheCys Ile Ser Lys Trp Tyr Leu Lys Glu Val 435 440 445 Lys Arg Lys Pro TyrAsn Leu Lys Leu Gly Cys Asn Trp Gln Phe Ile 450 455 460 Pro Lys Asp GluGly Trp Thr Glu 465 470 31 base pairs nucleic acid single linear othernucleic acid /desc = “DNA” 47 CGCCATGGCT GATACAATAC TAATAGAAAT G 31 29base pairs nucleic acid single linear other nucleic acid /desc = “DNA”48 GCAAGCTTTT ATTCAGTCCA CCCTTCATC 29 3753 base pairs nucleic aciddouble linear DNA (genomic) CDS 1..3750 49 ATG CCA ACA ATT AAT AGT TTTAAT TAT AAT GAT CCT GTT AAT AAT AGA 48 Met Pro Thr Ile Asn Ser Phe AsnTyr Asn Asp Pro Val Asn Asn Arg 1 5 10 15 ACA ATT TTA TAT ATT AAA CCAGGC GGT TGT CAA CAA TTT TAT AAA TCA 96 Thr Ile Leu Tyr Ile Lys Pro GlyGly Cys Gln Gln Phe Tyr Lys Ser 20 25 30 TTT AAT ATT ATG AAA AAT ATT TGGATA ATT CCA GAG AGA AAT GTA ATT 144 Phe Asn Ile Met Lys Asn Ile Trp IleIle Pro Glu Arg Asn Val Ile 35 40 45 GGT ACA ATT CCC CAA GAT TTT CTT CCGCCT ACT TCA TTG AAA AAT GGA 192 Gly Thr Ile Pro Gln Asp Phe Leu Pro ProThr Ser Leu Lys Asn Gly 50 55 60 GAT AGT AGT TAT TAT GAC CCT AAT TAT TTACAA AGT GAT CAA GAA AAG 240 Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu GlnSer Asp Gln Glu Lys 65 70 75 80 GAT AAA TTT TTA AAA ATA GTC ACA AAA ATATTT AAT AGA ATA AAT GAT 288 Asp Lys Phe Leu Lys Ile Val Thr Lys Ile PheAsn Arg Ile Asn Asp 85 90 95 AAT CTT TCA GGA AGG ATT TTA TTA GAA GAA CTGTCA AAA GCT AAT CCA 336 Asn Leu Ser Gly Arg Ile Leu Leu Glu Glu Leu SerLys Ala Asn Pro 100 105 110 TAT TTA GGA AAT GAT AAT ACT CCA GAT GGT GACTTC ATT ATT AAT GAT 384 Tyr Leu Gly Asn Asp Asn Thr Pro Asp Gly Asp PheIle Ile Asn Asp 115 120 125 GCA TCA GCA GTT CCA ATT CAA TTC TCA AAT GGTAGC CAA AGC ATA CTA 432 Ala Ser Ala Val Pro Ile Gln Phe Ser Asn Gly SerGln Ser Ile Leu 130 135 140 TTA CCT AAT GTT ATT ATA ATG GGA GCA GAG CCTGAT TTA TTT GAA ACT 480 Leu Pro Asn Val Ile Ile Met Gly Ala Glu Pro AspLeu Phe Glu Thr 145 150 155 160 AAC AGT TCC AAT ATT TCT CTA AGA AAT AATTAT ATG CCA AGC AAT CAC 528 Asn Ser Ser Asn Ile Ser Leu Arg Asn Asn TyrMet Pro Ser Asn His 165 170 175 GGT TTT GGA TCA ATA GCT ATA GTA ACA TTCTCA CCT GAA TAT TCT TTT 576 Gly Phe Gly Ser Ile Ala Ile Val Thr Phe SerPro Glu Tyr Ser Phe 180 185 190 AGA TTT AAA GAT AAT AGT ATG AAT GAA TTTATT CAA GAT CCT GCT CTT 624 Arg Phe Lys Asp Asn Ser Met Asn Glu Phe IleGln Asp Pro Ala Leu 195 200 205 ACA TTA ATG CAT GAA TTA ATA CAT TCA TTACAT GGA CTA TAT GGG GCT 672 Thr Leu Met His Glu Leu Ile His Ser Leu HisGly Leu Tyr Gly Ala 210 215 220 AAA GGG ATT ACT ACA AAG TAT ACT ATA ACACAA AAA CAA AAT CCC CTA 720 Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr GlnLys Gln Asn Pro Leu 225 230 235 240 ATA ACA AAT ATA AGA GGT ACA AAT ATTGAA GAA TTC TTA ACT TTT GGA 768 Ile Thr Asn Ile Arg Gly Thr Asn Ile GluGlu Phe Leu Thr Phe Gly 245 250 255 GGT ACT GAT TTA AAC ATT ATT ACT AGTGCT CAG TCC AAT GAT ATC TAT 816 Gly Thr Asp Leu Asn Ile Ile Thr Ser AlaGln Ser Asn Asp Ile Tyr 260 265 270 ACT AAT CTT CTA GCT GAT TAT AAA AAAATA GCG TCT AAA CTT AGC AAA 864 Thr Asn Leu Leu Ala Asp Tyr Lys Lys IleAla Ser Lys Leu Ser Lys 275 280 285 GTA CAA GTA TCT AAT CCA CTA CTT AATCCT TAT AAA GAT GTT TTT GAA 912 Val Gln Val Ser Asn Pro Leu Leu Asn ProTyr Lys Asp Val Phe Glu 290 295 300 GCA AAG TAT GGA TTA GAT AAA GAT GCTAGC GGA ATT TAT TCG GTA AAT 960 Ala Lys Tyr Gly Leu Asp Lys Asp Ala SerGly Ile Tyr Ser Val Asn 305 310 315 320 ATA AAC AAA TTT AAT GAT ATT TTTAAA AAA TTA TAC AGC TTT ACG GAA 1008 Ile Asn Lys Phe Asn Asp Ile Phe LysLys Leu Tyr Ser Phe Thr Glu 325 330 335 TTT GAT TTA GCA ACT AAA TTT CAAGTT AAA TGT AGG CAA ACT TAT ATT 1056 Phe Asp Leu Ala Thr Lys Phe Gln ValLys Cys Arg Gln Thr Tyr Ile 340 345 350 GGA CAG TAT AAA TAC TTC AAA CTTTCA AAC TTG TTA AAT GAT TCT ATT 1104 Gly Gln Tyr Lys Tyr Phe Lys Leu SerAsn Leu Leu Asn Asp Ser Ile 355 360 365 TAT AAT ATA TCA GAA GGC TAT AATATA AAT AAT TTA AAG GTA AAT TTT 1152 Tyr Asn Ile Ser Glu Gly Tyr Asn IleAsn Asn Leu Lys Val Asn Phe 370 375 380 AGA GGA CAG AAT GCA AAT TTA AATCCT AGA ATT ATT ACA CCA ATT ACA 1200 Arg Gly Gln Asn Ala Asn Leu Asn ProArg Ile Ile Thr Pro Ile Thr 385 390 395 400 GGT AGA GGA CTA GTA AAA AAAATC ATT AGA TTT TGT AAA AAT ATT GTT 1248 Gly Arg Gly Leu Val Lys Lys IleIle Arg Phe Cys Lys Asn Ile Val 405 410 415 TCT GTA AAA GGC ATA AGG AAATCA ATA TGT ATC GAA ATA AAT AAT GGT 1296 Ser Val Lys Gly Ile Arg Lys SerIle Cys Ile Glu Ile Asn Asn Gly 420 425 430 GAG TTA TTT TTT GTG GCT TCCGAG AAT AGT TAT AAT GAT GAT AAT ATA 1344 Glu Leu Phe Phe Val Ala Ser GluAsn Ser Tyr Asn Asp Asp Asn Ile 435 440 445 AAT ACT CCT AAA GAA ATT GACGAT ACA GTA ACT TCA AAT AAT AAT TAT 1392 Asn Thr Pro Lys Glu Ile Asp AspThr Val Thr Ser Asn Asn Asn Tyr 450 455 460 GAA AAT GAT TTA GAT CAG GTTATT TTA AAT TTT AAT AGT GAA TCA GCA 1440 Glu Asn Asp Leu Asp Gln Val IleLeu Asn Phe Asn Ser Glu Ser Ala 465 470 475 480 CCT GGA CTT TCA GAT GAAAAA TTA AAT TTA ACT ATC CAA AAT GAT GCT 1488 Pro Gly Leu Ser Asp Glu LysLeu Asn Leu Thr Ile Gln Asn Asp Ala 485 490 495 TAT ATA CCA AAA TAT GATTCT AAT GGA ACA AGT GAT ATA GAA CAA CAT 1536 Tyr Ile Pro Lys Tyr Asp SerAsn Gly Thr Ser Asp Ile Glu Gln His 500 505 510 GAT GTT AAT GAA CTT AATGTA TTT TTC TAT TTA GAT GCA CAG AAA GTG 1584 Asp Val Asn Glu Leu Asn ValPhe Phe Tyr Leu Asp Ala Gln Lys Val 515 520 525 CCC GAA GGT GAA AAT AATGTC AAT CTC ACC TCT TCA ATT GAT ACA GCA 1632 Pro Glu Gly Glu Asn Asn ValAsn Leu Thr Ser Ser Ile Asp Thr Ala 530 535 540 TTA TTA GAA CAA CCT AAAATA TAT ACA TTT TTT TCA TCA GAA TTT ATT 1680 Leu Leu Glu Gln Pro Lys IleTyr Thr Phe Phe Ser Ser Glu Phe Ile 545 550 555 560 AAT AAT GTC AAT AAACCT GTG CAA GCA GCA TTA TTT GTA AGC TGG ATA 1728 Asn Asn Val Asn Lys ProVal Gln Ala Ala Leu Phe Val Ser Trp Ile 565 570 575 CAA CAA GTA TTA GTAGAT TTT ACT ACT GAA GCT AAC CAA AAA AGT ACT 1776 Gln Gln Val Leu Val AspPhe Thr Thr Glu Ala Asn Gln Lys Ser Thr 580 585 590 GTT GAT AAA ATT GCAGAT ATT TCT ATA GTT GTT CCA TAT ATA GGT CTT 1824 Val Asp Lys Ile Ala AspIle Ser Ile Val Val Pro Tyr Ile Gly Leu 595 600 605 GCT TTA AAT ATA GGAAAT GAA GCA CAA AAA GGA AAT TTT AAA GAT GCA 1872 Ala Leu Asn Ile Gly AsnGlu Ala Gln Lys Gly Asn Phe Lys Asp Ala 610 615 620 CTT GAA TTA TTA GGAGCA GGT ATT TTA TTA GAA TTT GAA CCC GAG CTT 1920 Leu Glu Leu Leu Gly AlaGly Ile Leu Leu Glu Phe Glu Pro Glu Leu 625 630 635 640 TTA ATT CCT ACAATT TTA GTA TTC ACG ATA AAA TCT TTT TTA GGT TCA 1968 Leu Ile Pro Thr IleLeu Val Phe Thr Ile Lys Ser Phe Leu Gly Ser 645 650 655 TCT GAT AAT AAAAAT AAA GTT ATT AAA GCA ATA AAT AAT GCA TTG AAA 2016 Ser Asp Asn Lys AsnLys Val Ile Lys Ala Ile Asn Asn Ala Leu Lys 660 665 670 GAA AGA GAT GAAAAA TGG AAA GAA GTA TAT AGT TTT ATA GTA TCG AAT 2064 Glu Arg Asp Glu LysTrp Lys Glu Val Tyr Ser Phe Ile Val Ser Asn 675 680 685 TGG ATG ACT AAAATT AAT ACA CAA TTT AAT AAA AGA AAA GAA CAA ATG 2112 Trp Met Thr Lys IleAsn Thr Gln Phe Asn Lys Arg Lys Glu Gln Met 690 695 700 TAT CAA GCT TTACAA AAT CAA GTA AAT GCA CTT AAA GCA ATA ATA GAA 2160 Tyr Gln Ala Leu GlnAsn Gln Val Asn Ala Leu Lys Ala Ile Ile Glu 705 710 715 720 TCT AAG TATAAT AGT TAT ACT TTA GAA GAA AAA AAT GAG CTT ACA AAT 2208 Ser Lys Tyr AsnSer Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn 725 730 735 AAA TAT GATATT GAG CAA ATA GAA AAT GAA CTT AAT CAA AAG GTT TCT 2256 Lys Tyr Asp IleGlu Gln Ile Glu Asn Glu Leu Asn Gln Lys Val Ser 740 745 750 ATA GCA ATGAAT AAT ATA GAC AGG TTC TTA ACT GAA AGT TCT ATA TCT 2304 Ile Ala Met AsnAsn Ile Asp Arg Phe Leu Thr Glu Ser Ser Ile Ser 755 760 765 TAT TTA ATGAAA TTA ATA AAT GAA GTA AAA ATT AAT AAA TTA AGA GAA 2352 Tyr Leu Met LysLeu Ile Asn Glu Val Lys Ile Asn Lys Leu Arg Glu 770 775 780 TAT GAT GAAAAT GTT AAA ACG TAT TTA TTA GAT TAT ATT ATA AAA CAT 2400 Tyr Asp Glu AsnVal Lys Thr Tyr Leu Leu Asp Tyr Ile Ile Lys His 785 790 795 800 GGA TCAATC TTG GGA GAG AGT CAG CAA GAA CTA AAT TCT ATG GTA ATT 2448 Gly Ser IleLeu Gly Glu Ser Gln Gln Glu Leu Asn Ser Met Val Ile 805 810 815 GAT ACCCTA AAT AAT AGT ATT CCT TTT AAG CTT TCT TCT TAT ACA GAT 2496 Asp Thr LeuAsn Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp 820 825 830 GAT AAAATT TTA ATT TCA TAT TTT AAT AAG TTC TTT AAG AGA ATT AAA 2544 Asp Lys IleLeu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys 835 840 845 AGT AGTTCT GTT TTA AAT ATG AGA TAT AAA AAT GAT AAA TAC GTA GAT 2592 Ser Ser SerVal Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp 850 855 860 ACT TCAGGA TAT GAT TCA AAT ATA AAT ATT AAT GGA GAT GTA TAT AAA 2640 Thr Ser GlyTyr Asp Ser Asn Ile Asn Ile Asn Gly Asp Val Tyr Lys 865 870 875 880 TATCCA ACT AAT AAA AAT CAA TTT GGA ATA TAT AAT GAT AAA CTT AGT 2688 Tyr ProThr Asn Lys Asn Gln Phe Gly Ile Tyr Asn Asp Lys Leu Ser 885 890 895 GAAGTT AAT ATA TCT CAA AAT GAT TAC ATT ATA TAT GAT AAT AAA TAT 2736 Glu ValAsn Ile Ser Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr 900 905 910 AAAAAT TTT AGT ATT AGT TTT TGG GTA AGA ATT CCT AAC TAT GAT AAT 2784 Lys AsnPhe Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn 915 920 925 AAGATA GTA AAT GTT AAT AAT GAA TAC ACT ATA ATA AAT TGT ATG AGG 2832 Lys IleVal Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg 930 935 940 GATAAT AAT TCA GGA TGG AAA GTA TCT CTT AAT CAT AAT GAA ATA ATT 2880 Asp AsnAsn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile 945 950 955 960TGG ACA TTG CAA GAT AAT TCA GGA ATT AAT CAA AAA TTA GCA TTT AAC 2928 TrpThr Leu Gln Asp Asn Ser Gly Ile Asn Gln Lys Leu Ala Phe Asn 965 970 975TAT GGT AAC GCA AAT GGT ATT TCT GAT TAT ATA AAT AAG TGG ATT TTT 2976 TyrGly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe 980 985 990GTA ACT ATA ACT AAT GAT AGA TTA GGA GAT TCT AAA CTT TAT ATT AAT 3024 ValThr Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn 995 10001005 GGA AAT TTA ATA GAT AAA AAA TCA ATT TTA AAT TTA GGT AAT ATT CAT3072 Gly Asn Leu Ile Asp Lys Lys Ser Ile Leu Asn Leu Gly Asn Ile His1010 1015 1020 GTT AGT GAC AAT ATA TTA TTT AAA ATA GTT AAT TGT AGT TATACA AGA 3120 Val Ser Asp Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr ThrArg 1025 1030 1035 1040 TAT ATT GGT ATT AGA TAT TTT AAT ATT TTT GAT AAAGAA TTA GAT GAA 3168 Tyr Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp Lys GluLeu Asp Glu 1045 1050 1055 ACA GAA ATT CAA ACT TTA TAT AAC AAT GAA CCTAAT GCA AAT ATT TTA 3216 Thr Glu Ile Gln Thr Leu Tyr Asn Asn Glu Pro AsnAla Asn Ile Leu 1060 1065 1070 AAG GAT TTT TGG GGA AAT TAT TTG CTT TATGAC AAA GAA TAC TAT TTA 3264 Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr AspLys Glu Tyr Tyr Leu 1075 1080 1085 TTA AAT GTG TTA AAA CCA AAT AAC TTTATT AAT AGG AGA ACA GAT TCT 3312 Leu Asn Val Leu Lys Pro Asn Asn Phe IleAsn Arg Arg Thr Asp Ser 1090 1095 1100 ACT TTA AGC ATT AAT AAT ATA AGAAGC ACT ATT CTT TTA GCT AAT AGA 3360 Thr Leu Ser Ile Asn Asn Ile Arg SerThr Ile Leu Leu Ala Asn Arg 1105 1110 1115 1120 TTA TAT AGT GGA ATA AAAGTT AAA ATA CAA AGA GTT AAT AAT AGT AGT 3408 Leu Tyr Ser Gly Ile Lys ValLys Ile Gln Arg Val Asn Asn Ser Ser 1125 1130 1135 ACT AAC GAT AAT CTTGTT AGA AAG AAT GAT CAG GTA TAT ATT AAT TTT 3456 Thr Asn Asp Asn Leu ValArg Lys Asn Asp Gln Val Tyr Ile Asn Phe 1140 1145 1150 GTA GCC AGC AAAACT CAC TTA CTT CCA TTA TAT GCT GAT ACA GCT ACC 3504 Val Ala Ser Lys ThrHis Leu Leu Pro Leu Tyr Ala Asp Thr Ala Thr 1155 1160 1165 ACA AAT AAAGAG AAA ACA ATA AAA ATA TCA TCA TCT GGC AAT AGA TTT 3552 Thr Asn Lys GluLys Thr Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe 1170 1175 1180 AAT CAAGTA GTA GTT ATG AAT TCA GTA GGA TGT ACA ATG AAT TTT AAA 3600 Asn Gln ValVal Val Met Asn Ser Val Gly Cys Thr Met Asn Phe Lys 1185 1190 1195 1200AAT AAT AAT GGA AAT AAT ATT GGG TTG TTA GGT TTC AAG GCA GAT ACT 3648 AsnAsn Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala Asp Thr 1205 12101215 GTA GTT GCT AGT ACT TGG TAT TAT ACA CAT ATG AGA GAT AAT ACA AAC3696 Val Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp Asn Thr Asn1220 1225 1230 AGC AAT GGA TTT TTT TGG AAC TTT ATT TCT GAA GAA CAT GGATGG CAA 3744 Ser Asn Gly Phe Phe Trp Asn Phe Ile Ser Glu Glu His Gly TrpGln 1235 1240 1245 GAA AAA TAA 3753 Glu Lys 1250 1250 amino acids aminoacid linear protein 50 Met Pro Thr Ile Asn Ser Phe Asn Tyr Asn Asp ProVal Asn Asn Arg 1 5 10 15 Thr Ile Leu Tyr Ile Lys Pro Gly Gly Cys GlnGln Phe Tyr Lys Ser 20 25 30 Phe Asn Ile Met Lys Asn Ile Trp Ile Ile ProGlu Arg Asn Val Ile 35 40 45 Gly Thr Ile Pro Gln Asp Phe Leu Pro Pro ThrSer Leu Lys Asn Gly 50 55 60 Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu GlnSer Asp Gln Glu Lys 65 70 75 80 Asp Lys Phe Leu Lys Ile Val Thr Lys IlePhe Asn Arg Ile Asn Asp 85 90 95 Asn Leu Ser Gly Arg Ile Leu Leu Glu GluLeu Ser Lys Ala Asn Pro 100 105 110 Tyr Leu Gly Asn Asp Asn Thr Pro AspGly Asp Phe Ile Ile Asn Asp 115 120 125 Ala Ser Ala Val Pro Ile Gln PheSer Asn Gly Ser Gln Ser Ile Leu 130 135 140 Leu Pro Asn Val Ile Ile MetGly Ala Glu Pro Asp Leu Phe Glu Thr 145 150 155 160 Asn Ser Ser Asn IleSer Leu Arg Asn Asn Tyr Met Pro Ser Asn His 165 170 175 Gly Phe Gly SerIle Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe 180 185 190 Arg Phe LysAsp Asn Ser Met Asn Glu Phe Ile Gln Asp Pro Ala Leu 195 200 205 Thr LeuMet His Glu Leu Ile His Ser Leu His Gly Leu Tyr Gly Ala 210 215 220 LysGly Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys Gln Asn Pro Leu 225 230 235240 Ile Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Leu Thr Phe Gly 245250 255 Gly Thr Asp Leu Asn Ile Ile Thr Ser Ala Gln Ser Asn Asp Ile Tyr260 265 270 Thr Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser Lys Leu SerLys 275 280 285 Val Gln Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp ValPhe Glu 290 295 300 Ala Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile TyrSer Val Asn 305 310 315 320 Ile Asn Lys Phe Asn Asp Ile Phe Lys Lys LeuTyr Ser Phe Thr Glu 325 330 335 Phe Asp Leu Ala Thr Lys Phe Gln Val LysCys Arg Gln Thr Tyr Ile 340 345 350 Gly Gln Tyr Lys Tyr Phe Lys Leu SerAsn Leu Leu Asn Asp Ser Ile 355 360 365 Tyr Asn Ile Ser Glu Gly Tyr AsnIle Asn Asn Leu Lys Val Asn Phe 370 375 380 Arg Gly Gln Asn Ala Asn LeuAsn Pro Arg Ile Ile Thr Pro Ile Thr 385 390 395 400 Gly Arg Gly Leu ValLys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val 405 410 415 Ser Val Lys GlyIle Arg Lys Ser Ile Cys Ile Glu Ile Asn Asn Gly 420 425 430 Glu Leu PhePhe Val Ala Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile 435 440 445 Asn ThrPro Lys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr 450 455 460 GluAsn Asp Leu Asp Gln Val Ile Leu Asn Phe Asn Ser Glu Ser Ala 465 470 475480 Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr Ile Gln Asn Asp Ala 485490 495 Tyr Ile Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp Ile Glu Gln His500 505 510 Asp Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp Ala Gln LysVal 515 520 525 Pro Glu Gly Glu Asn Asn Val Asn Leu Thr Ser Ser Ile AspThr Ala 530 535 540 Leu Leu Glu Gln Pro Lys Ile Tyr Thr Phe Phe Ser SerGlu Phe Ile 545 550 555 560 Asn Asn Val Asn Lys Pro Val Gln Ala Ala LeuPhe Val Ser Trp Ile 565 570 575 Gln Gln Val Leu Val Asp Phe Thr Thr GluAla Asn Gln Lys Ser Thr 580 585 590 Val Asp Lys Ile Ala Asp Ile Ser IleVal Val Pro Tyr Ile Gly Leu 595 600 605 Ala Leu Asn Ile Gly Asn Glu AlaGln Lys Gly Asn Phe Lys Asp Ala 610 615 620 Leu Glu Leu Leu Gly Ala GlyIle Leu Leu Glu Phe Glu Pro Glu Leu 625 630 635 640 Leu Ile Pro Thr IleLeu Val Phe Thr Ile Lys Ser Phe Leu Gly Ser 645 650 655 Ser Asp Asn LysAsn Lys Val Ile Lys Ala Ile Asn Asn Ala Leu Lys 660 665 670 Glu Arg AspGlu Lys Trp Lys Glu Val Tyr Ser Phe Ile Val Ser Asn 675 680 685 Trp MetThr Lys Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu Gln Met 690 695 700 TyrGln Ala Leu Gln Asn Gln Val Asn Ala Leu Lys Ala Ile Ile Glu 705 710 715720 Ser Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn 725730 735 Lys Tyr Asp Ile Glu Gln Ile Glu Asn Glu Leu Asn Gln Lys Val Ser740 745 750 Ile Ala Met Asn Asn Ile Asp Arg Phe Leu Thr Glu Ser Ser IleSer 755 760 765 Tyr Leu Met Lys Leu Ile Asn Glu Val Lys Ile Asn Lys LeuArg Glu 770 775 780 Tyr Asp Glu Asn Val Lys Thr Tyr Leu Leu Asp Tyr IleIle Lys His 785 790 795 800 Gly Ser Ile Leu Gly Glu Ser Gln Gln Glu LeuAsn Ser Met Val Ile 805 810 815 Asp Thr Leu Asn Asn Ser Ile Pro Phe LysLeu Ser Ser Tyr Thr Asp 820 825 830 Asp Lys Ile Leu Ile Ser Tyr Phe AsnLys Phe Phe Lys Arg Ile Lys 835 840 845 Ser Ser Ser Val Leu Asn Met ArgTyr Lys Asn Asp Lys Tyr Val Asp 850 855 860 Thr Ser Gly Tyr Asp Ser AsnIle Asn Ile Asn Gly Asp Val Tyr Lys 865 870 875 880 Tyr Pro Thr Asn LysAsn Gln Phe Gly Ile Tyr Asn Asp Lys Leu Ser 885 890 895 Glu Val Asn IleSer Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr 900 905 910 Lys Asn PheSer Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn 915 920 925 Lys IleVal Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg 930 935 940 AspAsn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile 945 950 955960 Trp Thr Leu Gln Asp Asn Ser Gly Ile Asn Gln Lys Leu Ala Phe Asn 965970 975 Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe980 985 990 Val Thr Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr IleAsn 995 1000 1005 Gly Asn Leu Ile Asp Lys Lys Ser Ile Leu Asn Leu GlyAsn Ile His 1010 1015 1020 Val Ser Asp Asn Ile Leu Phe Lys Ile Val AsnCys Ser Tyr Thr Arg 1025 1030 1035 1040 Tyr Ile Gly Ile Arg Tyr Phe AsnIle Phe Asp Lys Glu Leu Asp Glu 1045 1050 1055 Thr Glu Ile Gln Thr LeuTyr Asn Asn Glu Pro Asn Ala Asn Ile Leu 1060 1065 1070 Lys Asp Phe TrpGly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu 1075 1080 1085 Leu AsnVal Leu Lys Pro Asn Asn Phe Ile Asn Arg Arg Thr Asp Ser 1090 1095 1100Thr Leu Ser Ile Asn Asn Ile Arg Ser Thr Ile Leu Leu Ala Asn Arg 11051110 1115 1120 Leu Tyr Ser Gly Ile Lys Val Lys Ile Gln Arg Val Asn AsnSer Ser 1125 1130 1135 Thr Asn Asp Asn Leu Val Arg Lys Asn Asp Gln ValTyr Ile Asn Phe 1140 1145 1150 Val Ala Ser Lys Thr His Leu Leu Pro LeuTyr Ala Asp Thr Ala Thr 1155 1160 1165 Thr Asn Lys Glu Lys Thr Ile LysIle Ser Ser Ser Gly Asn Arg Phe 1170 1175 1180 Asn Gln Val Val Val MetAsn Ser Val Gly Cys Thr Met Asn Phe Lys 1185 1190 1195 1200 Asn Asn AsnGly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala Asp Thr 1205 1210 1215 ValVal Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp Asn Thr Asn 1220 12251230 Ser Asn Gly Phe Phe Trp Asn Phe Ile Ser Glu Glu His Gly Trp Gln1235 1240 1245 Glu Lys 1250 3759 base pairs nucleic acid double linearDNA (genomic) CDS 1..3756 51 ATG CCA AAA ATT AAT AGT TTT AAT TAT AAT GATCCT GTT AAT GAT AGA 48 Met Pro Lys Ile Asn Ser Phe Asn Tyr Asn Asp ProVal Asn Asp Arg 1 5 10 15 ACA ATT TTA TAT ATT AAA CCA GGC GGT TGT CAAGAA TTT TAT AAA TCA 96 Thr Ile Leu Tyr Ile Lys Pro Gly Gly Cys Gln GluPhe Tyr Lys Ser 20 25 30 TTT AAT ATT ATG AAA AAT ATT TGG ATA ATT CCA GAGAGA AAT GTA ATT 144 Phe Asn Ile Met Lys Asn Ile Trp Ile Ile Pro Glu ArgAsn Val Ile 35 40 45 GGT ACA ACC CCC CAA GAT TTT CAT CCG CCT ACT TCA TTAAAA AAT GGA 192 Gly Thr Thr Pro Gln Asp Phe His Pro Pro Thr Ser Leu LysAsn Gly 50 55 60 GAT AGT AGT TAT TAT GAC CCT AAT TAT TTA CAA AGT GAT GAAGAA AAG 240 Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gln Ser Asp Glu GluLys 65 70 75 80 GAT AGA TTT TTA AAA ATA GTC ACA AAA ATA TTT AAT AGA ATAAAT AAT 288 Asp Arg Phe Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile AsnAsn 85 90 95 AAT CTT TCA GGA GGG ATT TTA TTA GAA GAA CTG TCA AAA GCT AATCCA 336 Asn Leu Ser Gly Gly Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro100 105 110 TAT TTA GGG AAT GAT AAT ACT CCA GAT AAT CAA TTC CAT ATT GGTGAT 384 Tyr Leu Gly Asn Asp Asn Thr Pro Asp Asn Gln Phe His Ile Gly Asp115 120 125 GCA TCA GCA GTT GAG ATT AAA TTC TCA AAT GGT AGC CAA GAC ATACTA 432 Ala Ser Ala Val Glu Ile Lys Phe Ser Asn Gly Ser Gln Asp Ile Leu130 135 140 TTA CCT AAT GTT ATT ATA ATG GGA GCA GAG CCT GAT TTA TTT GAAACT 480 Leu Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp Leu Phe Glu Thr145 150 155 160 AAC AGT TCC AAT ATT TCT CTA AGA AAT AAT TAT ATG CCA AGCAAT CAC 528 Asn Ser Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser AsnHis 165 170 175 GGT TTT GGA TCA ATA GCT ATA GTA ACA TTC TCA CCT GAA TATTCT TTT 576 Gly Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr SerPhe 180 185 190 AGA TTT AAT GAT AAT AGT ATG AAT GAA TTT ATT CAA GAT CCTGCT CTT 624 Arg Phe Asn Asp Asn Ser Met Asn Glu Phe Ile Gln Asp Pro AlaLeu 195 200 205 ACA TTA ATG CAT GAA TTA ATA CAT TCA TTA CAT GGA CTA TATGGG GCT 672 Thr Leu Met His Glu Leu Ile His Ser Leu His Gly Leu Tyr GlyAla 210 215 220 AAA GGG ATT ACT ACA AAG TAT ACT ATA ACA CAA AAA CAA AATCCC CTA 720 Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys Gln Asn ProLeu 225 230 235 240 ATA ACA AAT ATA AGA GGT ACA AAT ATT GAA GAA TTC TTAACT TTT GGA 768 Ile Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Leu ThrPhe Gly 245 250 255 GGT ACT GAT TTA AAC ATT ATT ACT AGT GCT CAG TCC AATGAT ATC TAT 816 Gly Thr Asp Leu Asn Ile Ile Thr Ser Ala Gln Ser Asn AspIle Tyr 260 265 270 ACT AAT CTT CTA GCT GAT TAT AAA AAA ATA GCG TCT AAACTT AGC AAA 864 Thr Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser Lys LeuSer Lys 275 280 285 GTA CAA GTA TCT AAT CCA CTA CTT AAT CCT TAT AAA GATGTT TTT GAA 912 Val Gln Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp ValPhe Glu 290 295 300 GCA AAG TAT GGA TTA GAT AAA GAT GCT AGC GGA ATT TATTCG GTA AAT 960 Ala Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile Tyr SerVal Asn 305 310 315 320 ATA AAC AAA TTT AAT GAT ATT TTT AAA AAA TTA TACAGC TTT ACG GAA 1008 Ile Asn Lys Phe Asn Asp Ile Phe Lys Lys Leu Tyr SerPhe Thr Glu 325 330 335 TTT GAT TTA GCA ACT AAA TTT CAA GTT AAA TGT AGGCAA ACT TAT ATT 1056 Phe Asp Leu Ala Thr Lys Phe Gln Val Lys Cys Arg GlnThr Tyr Ile 340 345 350 GGA CAG TAT AAA TAC TTC AAA CTT TCA AAC TTG TTAAAT GAT TCT ATT 1104 Gly Gln Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu AsnAsp Ser Ile 355 360 365 TAT AAT ATA TCA GAA GGC TAT AAT ATA AAT AAT TTAAAG GTA AAT TTT 1152 Tyr Asn Ile Ser Glu Gly Tyr Asn Ile Asn Asn Leu LysVal Asn Phe 370 375 380 AGA GGA CAG AAT GCA AAT TTA AAT CCT AGA ATT ATTACA CCA ATT ACA 1200 Arg Gly Gln Asn Ala Asn Leu Asn Pro Arg Ile Ile ThrPro Ile Thr 385 390 395 400 GGT AGA GGA CTA GTA AAA AAA ATC ATT AGA TTTTGT AAA AAT ATT GTT 1248 Gly Arg Gly Leu Val Lys Lys Ile Ile Arg Phe CysLys Asn Ile Val 405 410 415 TCT GTA AAA GGC ATA AGG AAA TCA ATA TGT ATCGAA ATA AAT AAT GGT 1296 Ser Val Lys Gly Ile Arg Lys Ser Ile Cys Ile GluIle Asn Asn Gly 420 425 430 GAG TTA TTT TTT GTG GCT TCC GAG AAT AGT TATAAT GAT GAT AAT ATA 1344 Glu Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr AsnAsp Asp Asn Ile 435 440 445 AAT ACT CCT AAA GAA ATT GAC GAT ACA GTA ACTTCA AAT AAT AAT TAT 1392 Asn Thr Pro Lys Glu Ile Asp Asp Thr Val Thr SerAsn Asn Asn Tyr 450 455 460 GAA AAT GAT TTA GAT CAG GTT ATT TTA AAT TTTAAT AGT GAA TCA GCA 1440 Glu Asn Asp Leu Asp Gln Val Ile Leu Asn Phe AsnSer Glu Ser Ala 465 470 475 480 CCT GGA CTT TCA GAT GAA AAA TTA AAT TTAACT ATC CAA AAT GAT GCT 1488 Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu ThrIle Gln Asn Asp Ala 485 490 495 TAT ATA CCA AAA TAT GAT TCT AAT GGA ACAAGT GAT ATA GAA CAA CAT 1536 Tyr Ile Pro Lys Tyr Asp Ser Asn Gly Thr SerAsp Ile Glu Gln His 500 505 510 GAT GTT AAT GAA CTT AAT GTA TTT TTC TATTTA GAT GCA CAG AAA GTG 1584 Asp Val Asn Glu Leu Asn Val Phe Phe Tyr LeuAsp Ala Gln Lys Val 515 520 525 CCC GAA GGT GAA AAT AAT GTC AAT CTC ACCTCT TCA ATT GAT ACA GCA 1632 Pro Glu Gly Glu Asn Asn Val Asn Leu Thr SerSer Ile Asp Thr Ala 530 535 540 TTA TTA GAA CAA CCT AAA ATA TAT ACA TTTTTT TCA TCA GAA TTT ATT 1680 Leu Leu Glu Gln Pro Lys Ile Tyr Thr Phe PheSer Ser Glu Phe Ile 545 550 555 560 AAT AAT GTC AAT AAA CCT GTG CAA GCAGCA TTA TTT GTA AGC TGG ATA 1728 Asn Asn Val Asn Lys Pro Val Gln Ala AlaLeu Phe Val Ser Trp Ile 565 570 575 CAA CAA GTG TTA GTA GAT TTT ACT ACTGAA GCT AAC CAA AAA AGT ACT 1776 Gln Gln Val Leu Val Asp Phe Thr Thr GluAla Asn Gln Lys Ser Thr 580 585 590 GTT GAT AAA ATT GCA GAT ATT TCT ATAGTT GTT CCA TAT ATA GGT CTT 1824 Val Asp Lys Ile Ala Asp Ile Ser Ile ValVal Pro Tyr Ile Gly Leu 595 600 605 GCT TTA AAT ATA GGA AAT GAA GCA CAAAAA GGA AAT TTT AAA GAT GCA 1872 Ala Leu Asn Ile Gly Asn Glu Ala Gln LysGly Asn Phe Lys Asp Ala 610 615 620 CTT GAA TTA TTA GGA GCA GGT ATT TTATTA GAA TTT GAA CCC GAG CTT 1920 Leu Glu Leu Leu Gly Ala Gly Ile Leu LeuGlu Phe Glu Pro Glu Leu 625 630 635 640 TTA ATT CCT ACA ATT TTA GTA TTCACG ATA AAA TCT TTT TTA GGT TCA 1968 Leu Ile Pro Thr Ile Leu Val Phe ThrIle Lys Ser Phe Leu Gly Ser 645 650 655 TCT GAT AAT AAA AAT AAA GTT ATTAAA GCA ATA AAT AAT GCA TTG AAA 2016 Ser Asp Asn Lys Asn Lys Val Ile LysAla Ile Asn Asn Ala Leu Lys 660 665 670 GAA AGA GAT GAA AAA TGG AAA GAAGTA TAT AGT TTT ATA GTA TCG AAT 2064 Glu Arg Asp Glu Lys Trp Lys Glu ValTyr Ser Phe Ile Val Ser Asn 675 680 685 TGG ATG ACT AAA ATT AAT ACA CAATTT AAT AAA AGA AAA GAA CAA ATG 2112 Trp Met Thr Lys Ile Asn Thr Gln PheAsn Lys Arg Lys Glu Gln Met 690 695 700 TAT CAA GCT TTA CAA AAT CAA GTAAAT GCA ATT AAA ACA ATA ATA GAA 2160 Tyr Gln Ala Leu Gln Asn Gln Val AsnAla Ile Lys Thr Ile Ile Glu 705 710 715 720 TCT AAG TAT AAT AGT TAT ACTTTA GAG GAA AAA AAT GAG CTT ACA AAT 2208 Ser Lys Tyr Asn Ser Tyr Thr LeuGlu Glu Lys Asn Glu Leu Thr Asn 725 730 735 AAA TAT GAT ATT AAG CAA ATAGAA AAT GAA CTT AAT CAA AAG GTT TCT 2256 Lys Tyr Asp Ile Lys Gln Ile GluAsn Glu Leu Asn Gln Lys Val Ser 740 745 750 ATA GCA ATG AAT AAT ATA GACAGG TTC TTA ACT GAA AGT TCT ATA TCC 2304 Ile Ala Met Asn Asn Ile Asp ArgPhe Leu Thr Glu Ser Ser Ile Ser 755 760 765 TAT TTA ATG AAA TTA ATA AATGAA GTA AAA ATT AAT AAA TTA AGA GAA 2352 Tyr Leu Met Lys Leu Ile Asn GluVal Lys Ile Asn Lys Leu Arg Glu 770 775 780 TAT GAT GAG AAT GTC AAA ACGTAT TTA TTG AAT TAT ATT ATA CAA CAT 2400 Tyr Asp Glu Asn Val Lys Thr TyrLeu Leu Asn Tyr Ile Ile Gln His 785 790 795 800 GGA TCA ATC TTG GGA GAGAGT CAG CAA GAA CTA AAT TCT ATG GTA ACT 2448 Gly Ser Ile Leu Gly Glu SerGln Gln Glu Leu Asn Ser Met Val Thr 805 810 815 GAT ACC CTA AAT AAT AGTATT CCT TTT AAG CTT TCT TCT TAT ACA GAT 2496 Asp Thr Leu Asn Asn Ser IlePro Phe Lys Leu Ser Ser Tyr Thr Asp 820 825 830 GAT AAA ATT TTA ATT TCATAT TTT AAT AAA TTC TTT AAG AGA ATT AAA 2544 Asp Lys Ile Leu Ile Ser TyrPhe Asn Lys Phe Phe Lys Arg Ile Lys 835 840 845 AGT AGT TCA GTT TTA AATATG AGA TAT AAA AAT GAT AAA TAC GTA GAT 2592 Ser Ser Ser Val Leu Asn MetArg Tyr Lys Asn Asp Lys Tyr Val Asp 850 855 860 ACT TCA GGA TAT GAT TCAAAT ATA AAT ATT AAT GGA GAT GTA TAT AAA 2640 Thr Ser Gly Tyr Asp Ser AsnIle Asn Ile Asn Gly Asp Val Tyr Lys 865 870 875 880 TAT CCA ACT AAT AAAAAT CAA TTT GGA ATA TAT AAT GAT AAA CTT AGT 2688 Tyr Pro Thr Asn Lys AsnGln Phe Gly Ile Tyr Asn Asp Lys Leu Ser 885 890 895 GAA GTT AAT ATA TCTCAA AAT GAT TAC ATT ATA TAT GAT AAT AAA TAT 2736 Glu Val Asn Ile Ser GlnAsn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr 900 905 910 AAA AAT TTT AGT ATTAGT TTT TGG GTA AGA ATT CCT AAC TAT GAT AAT 2784 Lys Asn Phe Ser Ile SerPhe Trp Val Arg Ile Pro Asn Tyr Asp Asn 915 920 925 AAG ATA GTA AAT GTTAAT AAT GAA TAC ACT ATA ATA AAT TGT ATG AGA 2832 Lys Ile Val Asn Val AsnAsn Glu Tyr Thr Ile Ile Asn Cys Met Arg 930 935 940 GAT AAT AAT TCA GGATGG AAA GTA TCT CTT AAT CAT AAT GAA ATA ATT 2880 Asp Asn Asn Ser Gly TrpLys Val Ser Leu Asn His Asn Glu Ile Ile 945 950 955 960 TGG ACA TTG CAAGAT AAT GCA GGA ATT AAT CAA AAA TTA GCA TTT AAC 2928 Trp Thr Leu Gln AspAsn Ala Gly Ile Asn Gln Lys Leu Ala Phe Asn 965 970 975 TAT GGT AAC GCAAAT GGT ATT TCT GAT TAT ATA AAT AAG TGG ATT TTT 2976 Tyr Gly Asn Ala AsnGly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe 980 985 990 GTA ACT ATA ACTAAT GAT AGA TTA GGA GAT TCT AAA CTT TAT ATT AAT 3024 Val Thr Ile Thr AsnAsp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn 995 1000 1005 GGA AAT TTAATA GAT CAA AAA TCA ATT TTA AAT TTA GGT AAT ATT CAT 3072 Gly Asn Leu IleAsp Gln Lys Ser Ile Leu Asn Leu Gly Asn Ile His 1010 1015 1020 GTT AGTGAC AAT ATA TTA TTT AAA ATA GTT AAT TGT AGT TAT ACA AGA 3120 Val Ser AspAsn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg 1025 1030 1035 1040TAT ATT GGT ATT AGA TAT TTT AAT ATT TTT GAT AAA GAA TTA GAT GAA 3168 TyrIle Gly Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu Asp Glu 1045 10501055 ACA GAA ATT CAA ACT TTA TAT AGC AAT GAA CCT AAT ACA AAT ATT TTG3216 Thr Glu Ile Gln Thr Leu Tyr Ser Asn Glu Pro Asn Thr Asn Ile Leu1060 1065 1070 AAG GAT TTT TGG GGA AAT TAT TTG CTT TAT GAC AAA GAA TACTAT TTA 3264 Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr TyrLeu 1075 1080 1085 TTA AAT GTG TTA AAA CCA AAT AAC TTT ATT GAT AGG AGAAAA GAT TCT 3312 Leu Asn Val Leu Lys Pro Asn Asn Phe Ile Asp Arg Arg LysAsp Ser 1090 1095 1100 ACT TTA AGC ATT AAT AAT ATA AGA AGC ACT ATT CTTTTA GCT AAT AGA 3360 Thr Leu Ser Ile Asn Asn Ile Arg Ser Thr Ile Leu LeuAla Asn Arg 1105 1110 1115 1120 TTA TAT AGT GGA ATA AAA GTT AAA ATA CAAAGA GTT AAT AAT AGT AGT 3408 Leu Tyr Ser Gly Ile Lys Val Lys Ile Gln ArgVal Asn Asn Ser Ser 1125 1130 1135 ACT AAC GAT AAT CTT GTT AGA AAG AATGAT CAG GTA TAT ATT AAT TTT 3456 Thr Asn Asp Asn Leu Val Arg Lys Asn AspGln Val Tyr Ile Asn Phe 1140 1145 1150 GTA GCC AGC AAA ACT CAC TTA TTTCCA TTA TAT GCT GAT ACA GCT ACC 3504 Val Ala Ser Lys Thr His Leu Phe ProLeu Tyr Ala Asp Thr Ala Thr 1155 1160 1165 ACA AAT AAA GAG AAA ACA ATAAAA ATA TCA TCA TCT GGC AAT AGA TTT 3552 Thr Asn Lys Glu Lys Thr Ile LysIle Ser Ser Ser Gly Asn Arg Phe 1170 1175 1180 AAT CAA GTA GTA GTT ATGAAT TCA GTA GGA AAT AAT TGT ACA ATG AAT 3600 Asn Gln Val Val Val Met AsnSer Val Gly Asn Asn Cys Thr Met Asn 1185 1190 1195 1200 TTT AAA AAT AATAAT GGA AAT AAT ATT GGG TTG TTA GGT TTC AAG GCA 3648 Phe Lys Asn Asn AsnGly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala 1205 1210 1215 GAT ACT GTAGTT GCT AGT ACT TGG TAT TAT ACA CAT ATG AGA GAT CAT 3696 Asp Thr Val ValAla Ser Thr Trp Tyr Tyr Thr His Met Arg Asp His 1220 1225 1230 ACA AACAGC AAT GGA TGT TTT TGG AAC TTT ATT TCT GAA GAA CAT GGA 3744 Thr Asn SerAsn Gly Cys Phe Trp Asn Phe Ile Ser Glu Glu His Gly 1235 1240 1245 TGGCAA GAA AAA TAA 3759 Trp Gln Glu Lys 1250 1252 amino acids amino acidlinear protein 52 Met Pro Lys Ile Asn Ser Phe Asn Tyr Asn Asp Pro ValAsn Asp Arg 1 5 10 15 Thr Ile Leu Tyr Ile Lys Pro Gly Gly Cys Gln GluPhe Tyr Lys Ser 20 25 30 Phe Asn Ile Met Lys Asn Ile Trp Ile Ile Pro GluArg Asn Val Ile 35 40 45 Gly Thr Thr Pro Gln Asp Phe His Pro Pro Thr SerLeu Lys Asn Gly 50 55 60 Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gln SerAsp Glu Glu Lys 65 70 75 80 Asp Arg Phe Leu Lys Ile Val Thr Lys Ile PheAsn Arg Ile Asn Asn 85 90 95 Asn Leu Ser Gly Gly Ile Leu Leu Glu Glu LeuSer Lys Ala Asn Pro 100 105 110 Tyr Leu Gly Asn Asp Asn Thr Pro Asp AsnGln Phe His Ile Gly Asp 115 120 125 Ala Ser Ala Val Glu Ile Lys Phe SerAsn Gly Ser Gln Asp Ile Leu 130 135 140 Leu Pro Asn Val Ile Ile Met GlyAla Glu Pro Asp Leu Phe Glu Thr 145 150 155 160 Asn Ser Ser Asn Ile SerLeu Arg Asn Asn Tyr Met Pro Ser Asn His 165 170 175 Gly Phe Gly Ser IleAla Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe 180 185 190 Arg Phe Asn AspAsn Ser Met Asn Glu Phe Ile Gln Asp Pro Ala Leu 195 200 205 Thr Leu MetHis Glu Leu Ile His Ser Leu His Gly Leu Tyr Gly Ala 210 215 220 Lys GlyIle Thr Thr Lys Tyr Thr Ile Thr Gln Lys Gln Asn Pro Leu 225 230 235 240Ile Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Leu Thr Phe Gly 245 250255 Gly Thr Asp Leu Asn Ile Ile Thr Ser Ala Gln Ser Asn Asp Ile Tyr 260265 270 Thr Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser Lys Leu Ser Lys275 280 285 Val Gln Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val PheGlu 290 295 300 Ala Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile Tyr SerVal Asn 305 310 315 320 Ile Asn Lys Phe Asn Asp Ile Phe Lys Lys Leu TyrSer Phe Thr Glu 325 330 335 Phe Asp Leu Ala Thr Lys Phe Gln Val Lys CysArg Gln Thr Tyr Ile 340 345 350 Gly Gln Tyr Lys Tyr Phe Lys Leu Ser AsnLeu Leu Asn Asp Ser Ile 355 360 365 Tyr Asn Ile Ser Glu Gly Tyr Asn IleAsn Asn Leu Lys Val Asn Phe 370 375 380 Arg Gly Gln Asn Ala Asn Leu AsnPro Arg Ile Ile Thr Pro Ile Thr 385 390 395 400 Gly Arg Gly Leu Val LysLys Ile Ile Arg Phe Cys Lys Asn Ile Val 405 410 415 Ser Val Lys Gly IleArg Lys Ser Ile Cys Ile Glu Ile Asn Asn Gly 420 425 430 Glu Leu Phe PheVal Ala Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile 435 440 445 Asn Thr ProLys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr 450 455 460 Glu AsnAsp Leu Asp Gln Val Ile Leu Asn Phe Asn Ser Glu Ser Ala 465 470 475 480Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr Ile Gln Asn Asp Ala 485 490495 Tyr Ile Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp Ile Glu Gln His 500505 510 Asp Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp Ala Gln Lys Val515 520 525 Pro Glu Gly Glu Asn Asn Val Asn Leu Thr Ser Ser Ile Asp ThrAla 530 535 540 Leu Leu Glu Gln Pro Lys Ile Tyr Thr Phe Phe Ser Ser GluPhe Ile 545 550 555 560 Asn Asn Val Asn Lys Pro Val Gln Ala Ala Leu PheVal Ser Trp Ile 565 570 575 Gln Gln Val Leu Val Asp Phe Thr Thr Glu AlaAsn Gln Lys Ser Thr 580 585 590 Val Asp Lys Ile Ala Asp Ile Ser Ile ValVal Pro Tyr Ile Gly Leu 595 600 605 Ala Leu Asn Ile Gly Asn Glu Ala GlnLys Gly Asn Phe Lys Asp Ala 610 615 620 Leu Glu Leu Leu Gly Ala Gly IleLeu Leu Glu Phe Glu Pro Glu Leu 625 630 635 640 Leu Ile Pro Thr Ile LeuVal Phe Thr Ile Lys Ser Phe Leu Gly Ser 645 650 655 Ser Asp Asn Lys AsnLys Val Ile Lys Ala Ile Asn Asn Ala Leu Lys 660 665 670 Glu Arg Asp GluLys Trp Lys Glu Val Tyr Ser Phe Ile Val Ser Asn 675 680 685 Trp Met ThrLys Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu Gln Met 690 695 700 Tyr GlnAla Leu Gln Asn Gln Val Asn Ala Ile Lys Thr Ile Ile Glu 705 710 715 720Ser Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn 725 730735 Lys Tyr Asp Ile Lys Gln Ile Glu Asn Glu Leu Asn Gln Lys Val Ser 740745 750 Ile Ala Met Asn Asn Ile Asp Arg Phe Leu Thr Glu Ser Ser Ile Ser755 760 765 Tyr Leu Met Lys Leu Ile Asn Glu Val Lys Ile Asn Lys Leu ArgGlu 770 775 780 Tyr Asp Glu Asn Val Lys Thr Tyr Leu Leu Asn Tyr Ile IleGln His 785 790 795 800 Gly Ser Ile Leu Gly Glu Ser Gln Gln Glu Leu AsnSer Met Val Thr 805 810 815 Asp Thr Leu Asn Asn Ser Ile Pro Phe Lys LeuSer Ser Tyr Thr Asp 820 825 830 Asp Lys Ile Leu Ile Ser Tyr Phe Asn LysPhe Phe Lys Arg Ile Lys 835 840 845 Ser Ser Ser Val Leu Asn Met Arg TyrLys Asn Asp Lys Tyr Val Asp 850 855 860 Thr Ser Gly Tyr Asp Ser Asn IleAsn Ile Asn Gly Asp Val Tyr Lys 865 870 875 880 Tyr Pro Thr Asn Lys AsnGln Phe Gly Ile Tyr Asn Asp Lys Leu Ser 885 890 895 Glu Val Asn Ile SerGln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr 900 905 910 Lys Asn Phe SerIle Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn 915 920 925 Lys Ile ValAsn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg 930 935 940 Asp AsnAsn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile 945 950 955 960Trp Thr Leu Gln Asp Asn Ala Gly Ile Asn Gln Lys Leu Ala Phe Asn 965 970975 Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe 980985 990 Val Thr Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn995 1000 1005 Gly Asn Leu Ile Asp Gln Lys Ser Ile Leu Asn Leu Gly AsnIle His 1010 1015 1020 Val Ser Asp Asn Ile Leu Phe Lys Ile Val Asn CysSer Tyr Thr Arg 1025 1030 1035 1040 Tyr Ile Gly Ile Arg Tyr Phe Asn IlePhe Asp Lys Glu Leu Asp Glu 1045 1050 1055 Thr Glu Ile Gln Thr Leu TyrSer Asn Glu Pro Asn Thr Asn Ile Leu 1060 1065 1070 Lys Asp Phe Trp GlyAsn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu 1075 1080 1085 Leu Asn ValLeu Lys Pro Asn Asn Phe Ile Asp Arg Arg Lys Asp Ser 1090 1095 1100 ThrLeu Ser Ile Asn Asn Ile Arg Ser Thr Ile Leu Leu Ala Asn Arg 1105 11101115 1120 Leu Tyr Ser Gly Ile Lys Val Lys Ile Gln Arg Val Asn Asn SerSer 1125 1130 1135 Thr Asn Asp Asn Leu Val Arg Lys Asn Asp Gln Val TyrIle Asn Phe 1140 1145 1150 Val Ala Ser Lys Thr His Leu Phe Pro Leu TyrAla Asp Thr Ala Thr 1155 1160 1165 Thr Asn Lys Glu Lys Thr Ile Lys IleSer Ser Ser Gly Asn Arg Phe 1170 1175 1180 Asn Gln Val Val Val Met AsnSer Val Gly Asn Asn Cys Thr Met Asn 1185 1190 1195 1200 Phe Lys Asn AsnAsn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala 1205 1210 1215 Asp ThrVal Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp His 1220 1225 1230Thr Asn Ser Asn Gly Cys Phe Trp Asn Phe Ile Ser Glu Glu His Gly 12351240 1245 Trp Gln Glu Lys 1250 1463 base pairs nucleic acid doublelinear other nucleic acid /desc = “DNA” CDS 108..1460 53 AGATCTCGATCCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60 TTCCCCTCTAGAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACC ATG GGC CAT 116 Met Gly His 1CAT CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT ATC GAA GGT 164 HisHis His His His His His His His Ser Ser Gly His Ile Glu Gly 5 10 15 CGTCAT ATG GCT AGC ATG GCT CTT TCT TCT TAT ACA GAT GAT AAA ATT 212 Arg HisMet Ala Ser Met Ala Leu Ser Ser Tyr Thr Asp Asp Lys Ile 20 25 30 35 TTAATT TCA TAT TTT AAT AAG TTC TTT AAG AGA ATT AAA AGT AGT TCT 260 Leu IleSer Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys Ser Ser Ser 40 45 50 GTT TTAAAT ATG AGA TAT AAA AAT GAT AAA TAC GTA GAT ACT TCA GGA 308 Val Leu AsnMet Arg Tyr Lys Asn Asp Lys Tyr Val Asp Thr Ser Gly 55 60 65 TAT GAT TCAAAT ATA AAT ATT AAT GGA GAT GTA TAT AAA TAT CCA ACT 356 Tyr Asp Ser AsnIle Asn Ile Asn Gly Asp Val Tyr Lys Tyr Pro Thr 70 75 80 AAT AAA AAT CAATTT GGA ATA TAT AAT GAT AAA CTT AGT GAA GTT AAT 404 Asn Lys Asn Gln PheGly Ile Tyr Asn Asp Lys Leu Ser Glu Val Asn 85 90 95 ATA TCT CAA AAT GATTAC ATT ATA TAT GAT AAT AAA TAT AAA AAT TTT 452 Ile Ser Gln Asn Asp TyrIle Ile Tyr Asp Asn Lys Tyr Lys Asn Phe 100 105 110 115 AGT ATT AGT TTTTGG GTA AGA ATT CCT AAC TAT GAT AAT AAG ATA GTA 500 Ser Ile Ser Phe TrpVal Arg Ile Pro Asn Tyr Asp Asn Lys Ile Val 120 125 130 AAT GTT AAT AATGAA TAC ACT ATA ATA AAT TGT ATG AGG GAT AAT AAT 548 Asn Val Asn Asn GluTyr Thr Ile Ile Asn Cys Met Arg Asp Asn Asn 135 140 145 TCA GGA TGG AAAGTA TCT CTT AAT CAT AAT GAA ATA ATT TGG ACA TTG 596 Ser Gly Trp Lys ValSer Leu Asn His Asn Glu Ile Ile Trp Thr Leu 150 155 160 CAA GAT AAT TCAGGA ATT AAT CAA AAA TTA GCA TTT AAC TAT GGT AAC 644 Gln Asp Asn Ser GlyIle Asn Gln Lys Leu Ala Phe Asn Tyr Gly Asn 165 170 175 GCA AAT GGT ATTTCT GAT TAT ATA AAT AAG TGG ATT TTT GTA ACT ATA 692 Ala Asn Gly Ile SerAsp Tyr Ile Asn Lys Trp Ile Phe Val Thr Ile 180 185 190 195 ACT AAT GATAGA TTA GGA GAT TCT AAA CTT TAT ATT AAT GGA AAT TTA 740 Thr Asn Asp ArgLeu Gly Asp Ser Lys Leu Tyr Ile Asn Gly Asn Leu 200 205 210 ATA GAT AAAAAA TCA ATT TTA AAT TTA GGT AAT ATT CAT GTT AGT GAC 788 Ile Asp Lys LysSer Ile Leu Asn Leu Gly Asn Ile His Val Ser Asp 215 220 225 AAT ATA TTATTT AAA ATA GTT AAT TGT AGT TAT ACA AGA TAT ATT GGT 836 Asn Ile Leu PheLys Ile Val Asn Cys Ser Tyr Thr Arg Tyr Ile Gly 230 235 240 ATT AGA TATTTT AAT ATT TTT GAT AAA GAA TTA GAT GAA ACA GAA ATT 884 Ile Arg Tyr PheAsn Ile Phe Asp Lys Glu Leu Asp Glu Thr Glu Ile 245 250 255 CAA ACT TTATAT AAC AAT GAA CCT AAT GCA AAT ATT TTA AAG GAT TTT 932 Gln Thr Leu TyrAsn Asn Glu Pro Asn Ala Asn Ile Leu Lys Asp Phe 260 265 270 275 TGG GGAAAT TAT TTG CTT TAT GAC AAA GAA TAC TAT TTA TTA AAT GTG 980 Trp Gly AsnTyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu Leu Asn Val 280 285 290 TTA AAACCA AAT AAC TTT ATT AAT AGG AGA ACA GAT TCT ACT TTA AGC 1028 Leu Lys ProAsn Asn Phe Ile Asn Arg Arg Thr Asp Ser Thr Leu Ser 295 300 305 ATT AATAAT ATA AGA AGC ACT ATT CTT TTA GCT AAT AGA TTA TAT AGT 1076 Ile Asn AsnIle Arg Ser Thr Ile Leu Leu Ala Asn Arg Leu Tyr Ser 310 315 320 GGA ATAAAA GTT AAA ATA CAA AGA GTT AAT AAT AGT AGT ACT AAC GAT 1124 Gly Ile LysVal Lys Ile Gln Arg Val Asn Asn Ser Ser Thr Asn Asp 325 330 335 AAT CTTGTT AGA AAG AAT GAT CAG GTA TAT ATT AAT TTT GTA GCC AGC 1172 Asn Leu ValArg Lys Asn Asp Gln Val Tyr Ile Asn Phe Val Ala Ser 340 345 350 355 AAAACT CAC TTA CTT CCA TTA TAT GCT GAT ACA GCT ACC ACA AAT AAA 1220 Lys ThrHis Leu Leu Pro Leu Tyr Ala Asp Thr Ala Thr Thr Asn Lys 360 365 370 GAGAAA ACA ATA AAA ATA TCA TCA TCT GGC AAT AGA TTT AAT CAA GTA 1268 Glu LysThr Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe Asn Gln Val 375 380 385 GTAGTT ATG AAT TCA GTA GGA AAT TGT ACA ATG AAT TTT AAA AAT AAT 1316 Val ValMet Asn Ser Val Gly Asn Cys Thr Met Asn Phe Lys Asn Asn 390 395 400 AATGGA AAT AAT ATT GGG TTG TTA GGT TTC AAG GCA GAT ACT GTA GTT 1364 Asn GlyAsn Asn Ile Gly Leu Leu Gly Phe Lys Ala Asp Thr Val Val 405 410 415 GCTAGT ACT TGG TAT TAT ACA CAT ATG AGA GAT AAT ACA AAC AGC AAT 1412 Ala SerThr Trp Tyr Tyr Thr His Met Arg Asp Asn Thr Asn Ser Asn 420 425 430 435GGA TTT TTT TGG AAC TTT ATT TCT GAA GAA CAT GGA TGG CAA GAA AAA 1460 GlyPhe Phe Trp Asn Phe Ile Ser Glu Glu His Gly Trp Gln Glu Lys 440 445 450TAA 1463 451 amino acids amino acid linear protein 54 Met Gly His HisHis His His His His His His His Ser Ser Gly His 1 5 10 15 Ile Glu GlyArg His Met Ala Ser Met Ala Leu Ser Ser Tyr Thr Asp 20 25 30 Asp Lys IleLeu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys 35 40 45 Ser Ser SerVal Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp 50 55 60 Thr Ser GlyTyr Asp Ser Asn Ile Asn Ile Asn Gly Asp Val Tyr Lys 65 70 75 80 Tyr ProThr Asn Lys Asn Gln Phe Gly Ile Tyr Asn Asp Lys Leu Ser 85 90 95 Glu ValAsn Ile Ser Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr 100 105 110 LysAsn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn 115 120 125Lys Ile Val Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg 130 135140 Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile 145150 155 160 Trp Thr Leu Gln Asp Asn Ser Gly Ile Asn Gln Lys Leu Ala PheAsn 165 170 175 Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys TrpIle Phe 180 185 190 Val Thr Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys LeuTyr Ile Asn 195 200 205 Gly Asn Leu Ile Asp Lys Lys Ser Ile Leu Asn LeuGly Asn Ile His 210 215 220 Val Ser Asp Asn Ile Leu Phe Lys Ile Val AsnCys Ser Tyr Thr Arg 225 230 235 240 Tyr Ile Gly Ile Arg Tyr Phe Asn IlePhe Asp Lys Glu Leu Asp Glu 245 250 255 Thr Glu Ile Gln Thr Leu Tyr AsnAsn Glu Pro Asn Ala Asn Ile Leu 260 265 270 Lys Asp Phe Trp Gly Asn TyrLeu Leu Tyr Asp Lys Glu Tyr Tyr Leu 275 280 285 Leu Asn Val Leu Lys ProAsn Asn Phe Ile Asn Arg Arg Thr Asp Ser 290 295 300 Thr Leu Ser Ile AsnAsn Ile Arg Ser Thr Ile Leu Leu Ala Asn Arg 305 310 315 320 Leu Tyr SerGly Ile Lys Val Lys Ile Gln Arg Val Asn Asn Ser Ser 325 330 335 Thr AsnAsp Asn Leu Val Arg Lys Asn Asp Gln Val Tyr Ile Asn Phe 340 345 350 ValAla Ser Lys Thr His Leu Leu Pro Leu Tyr Ala Asp Thr Ala Thr 355 360 365Thr Asn Lys Glu Lys Thr Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe 370 375380 Asn Gln Val Val Val Met Asn Ser Val Gly Asn Cys Thr Met Asn Phe 385390 395 400 Lys Asn Asn Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys AlaAsp 405 410 415 Thr Val Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg AspAsn Thr 420 425 430 Asn Ser Asn Gly Phe Phe Trp Asn Phe Ile Ser Glu GluHis Gly Trp 435 440 445 Gln Glu Lys 450 1472 base pairs nucleic aciddouble linear other nucleic acid /desc = “DNA” CDS 108..1463 55AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACC ATG GGC CAT 116 MetGly His 1 CAT CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT ATC GAAGGT 164 His His His His His His His His His Ser Ser Gly His Ile Glu Gly5 10 15 CGT CAT ATG GCT AGC ATG GCT CTT TCT TCT TAT ACA GAT GAT AAA ATT212 Arg His Met Ala Ser Met Ala Leu Ser Ser Tyr Thr Asp Asp Lys Ile 2025 30 35 TTA ATT TCA TAT TTT AAT AAA TTC TTT AAG AGA ATT AAA AGT AGT TCA260 Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys Ser Ser Ser 4045 50 GTT TTA AAT ATG AGA TAT AAA AAT GAT AAA TAC GTA GAT ACT TCA GGA308 Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp Thr Ser Gly 5560 65 TAT GAT TCA AAT ATA AAT ATT AAT GGA GAT GTA TAT AAA TAT CCA ACT356 Tyr Asp Ser Asn Ile Asn Ile Asn Gly Asp Val Tyr Lys Tyr Pro Thr 7075 80 AAT AAA AAT CAA TTT GGA ATA TAT AAT GAT AAA CTT AGT GAA GTT AAT404 Asn Lys Asn Gln Phe Gly Ile Tyr Asn Asp Lys Leu Ser Glu Val Asn 8590 95 ATA TCT CAA AAT GAT TAC ATT ATA TAT GAT AAT AAA TAT AAA AAT TTT452 Ile Ser Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr Lys Asn Phe 100105 110 115 AGT ATT AGT TTT TGG GTA AGA ATT CCT AAC TAT GAT AAT AAG ATAGTA 500 Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn Lys Ile Val120 125 130 AAT GTT AAT AAT GAA TAC ACT ATA ATA AAT TGT ATG AGA GAT AATAAT 548 Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg Asp Asn Asn135 140 145 TCA GGA TGG AAA GTA TCT CTT AAT CAT AAT GAA ATA ATT TGG ACATTG 596 Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile Trp Thr Leu150 155 160 CAA GAT AAT GCA GGA ATT AAT CAA AAA TTA GCA TTT AAC TAT GGTAAC 644 Gln Asp Asn Ala Gly Ile Asn Gln Lys Leu Ala Phe Asn Tyr Gly Asn165 170 175 GCA AAT GGT ATT TCT GAT TAT ATA AAT AAG TGG ATT TTT GTA ACTATA 692 Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe Val Thr Ile180 185 190 195 ACT AAT GAT AGA TTA GGA GAT TCT AAA CTT TAT ATT AAT GGAAAT TTA 740 Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn Gly AsnLeu 200 205 210 ATA GAT CAA AAA TCA ATT TTA AAT TTA GGT AAT ATT CAT GTTAGT GAC 788 Ile Asp Gln Lys Ser Ile Leu Asn Leu Gly Asn Ile His Val SerAsp 215 220 225 AAT ATA TTA TTT AAA ATA GTT AAT TGT AGT TAT ACA AGA TATATT GGT 836 Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg Tyr IleGly 230 235 240 ATT AGA TAT TTT AAT ATT TTT GAT AAA GAA TTA GAT GAA ACAGAA ATT 884 Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu Asp Glu Thr GluIle 245 250 255 CAA ACT TTA TAT AGC AAT GAA CCT AAT ACA AAT ATT TTG AAGGAT TTT 932 Gln Thr Leu Tyr Ser Asn Glu Pro Asn Thr Asn Ile Leu Lys AspPhe 260 265 270 275 TGG GGA AAT TAT TTG CTT TAT GAC AAA GAA TAC TAT TTATTA AAT GTG 980 Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu LeuAsn Val 280 285 290 TTA AAA CCA AAT AAC TTT ATT GAT AGG AGA AAA GAT TCTACT TTA AGC 1028 Leu Lys Pro Asn Asn Phe Ile Asp Arg Arg Lys Asp Ser ThrLeu Ser 295 300 305 ATT AAT AAT ATA AGA AGC ACT ATT CTT TTA GCT AAT AGATTA TAT AGT 1076 Ile Asn Asn Ile Arg Ser Thr Ile Leu Leu Ala Asn Arg LeuTyr Ser 310 315 320 GGA ATA AAA GTT AAA ATA CAA AGA GTT AAT AAT AGT AGTACT AAC GAT 1124 Gly Ile Lys Val Lys Ile Gln Arg Val Asn Asn Ser Ser ThrAsn Asp 325 330 335 AAT CTT GTT AGA AAG AAT GAT CAG GTA TAT ATT AAT TTTGTA GCC AGC 1172 Asn Leu Val Arg Lys Asn Asp Gln Val Tyr Ile Asn Phe ValAla Ser 340 345 350 355 AAA ACT CAC TTA TTT CCA TTA TAT GCT GAT ACA GCTACC ACA AAT AAA 1220 Lys Thr His Leu Phe Pro Leu Tyr Ala Asp Thr Ala ThrThr Asn Lys 360 365 370 GAG AAA ACA ATA AAA ATA TCA TCA TCT GGC AAT AGATTT AAT CAA GTA 1268 Glu Lys Thr Ile Lys Ile Ser Ser Ser Gly Asn Arg PheAsn Gln Val 375 380 385 GTA GTT ATG AAT TCA GTA GGA AAT AAT TGT ACA ATGAAT TTT AAA AAT 1316 Val Val Met Asn Ser Val Gly Asn Asn Cys Thr Met AsnPhe Lys Asn 390 395 400 AAT AAT GGA AAT AAT ATT GGG TTG TTA GGT TTC AAGGCA GAT ACT GTA 1364 Asn Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys AlaAsp Thr Val 405 410 415 GTT GCT AGT ACT TGG TAT TAT ACA CAT ATG AGA GATCAT ACA AAC AGC 1412 Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp HisThr Asn Ser 420 425 430 435 AAT GGA TGT TTT TGG AAC TTT ATT TCT GAA GAACAT GGA TGG CAA GAA 1460 Asn Gly Cys Phe Trp Asn Phe Ile Ser Glu Glu HisGly Trp Gln Glu 440 445 450 AAA TAAAAGCTT 1472 Lys 452 amino acids aminoacid linear protein 56 Met Gly His His His His His His His His His HisSer Ser Gly His 1 5 10 15 Ile Glu Gly Arg His Met Ala Ser Met Ala LeuSer Ser Tyr Thr Asp 20 25 30 Asp Lys Ile Leu Ile Ser Tyr Phe Asn Lys PhePhe Lys Arg Ile Lys 35 40 45 Ser Ser Ser Val Leu Asn Met Arg Tyr Lys AsnAsp Lys Tyr Val Asp 50 55 60 Thr Ser Gly Tyr Asp Ser Asn Ile Asn Ile AsnGly Asp Val Tyr Lys 65 70 75 80 Tyr Pro Thr Asn Lys Asn Gln Phe Gly IleTyr Asn Asp Lys Leu Ser 85 90 95 Glu Val Asn Ile Ser Gln Asn Asp Tyr IleIle Tyr Asp Asn Lys Tyr 100 105 110 Lys Asn Phe Ser Ile Ser Phe Trp ValArg Ile Pro Asn Tyr Asp Asn 115 120 125 Lys Ile Val Asn Val Asn Asn GluTyr Thr Ile Ile Asn Cys Met Arg 130 135 140 Asp Asn Asn Ser Gly Trp LysVal Ser Leu Asn His Asn Glu Ile Ile 145 150 155 160 Trp Thr Leu Gln AspAsn Ala Gly Ile Asn Gln Lys Leu Ala Phe Asn 165 170 175 Tyr Gly Asn AlaAsn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe 180 185 190 Val Thr IleThr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn 195 200 205 Gly AsnLeu Ile Asp Gln Lys Ser Ile Leu Asn Leu Gly Asn Ile His 210 215 220 ValSer Asp Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg 225 230 235240 Tyr Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu Asp Glu 245250 255 Thr Glu Ile Gln Thr Leu Tyr Ser Asn Glu Pro Asn Thr Asn Ile Leu260 265 270 Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr TyrLeu 275 280 285 Leu Asn Val Leu Lys Pro Asn Asn Phe Ile Asp Arg Arg LysAsp Ser 290 295 300 Thr Leu Ser Ile Asn Asn Ile Arg Ser Thr Ile Leu LeuAla Asn Arg 305 310 315 320 Leu Tyr Ser Gly Ile Lys Val Lys Ile Gln ArgVal Asn Asn Ser Ser 325 330 335 Thr Asn Asp Asn Leu Val Arg Lys Asn AspGln Val Tyr Ile Asn Phe 340 345 350 Val Ala Ser Lys Thr His Leu Phe ProLeu Tyr Ala Asp Thr Ala Thr 355 360 365 Thr Asn Lys Glu Lys Thr Ile LysIle Ser Ser Ser Gly Asn Arg Phe 370 375 380 Asn Gln Val Val Val Met AsnSer Val Gly Asn Asn Cys Thr Met Asn 385 390 395 400 Phe Lys Asn Asn AsnGly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala 405 410 415 Asp Thr Val ValAla Ser Thr Trp Tyr Tyr Thr His Met Arg Asp His 420 425 430 Thr Asn SerAsn Gly Cys Phe Trp Asn Phe Ile Ser Glu Glu His Gly 435 440 445 Trp GlnGlu Lys 450 31 base pairs nucleic acid single linear other nucleic acid/desc = “DNA” 57 CGCCATGGCT CTTTCTTCTT ATACAGATGA T 31 29 base pairsnucleic acid single linear other nucleic acid /desc = “DNA” 58GCAAGCTTTT ATTTTTCTTG CCATCCATG 29 3876 base pairs nucleic acid doublelinear DNA (genomic) CDS 1..3873 59 ATG CCA ATA ACA ATT AAC AAC TTT AATTAT TCA GAT CCT GTT GAT AAT 48 Met Pro Ile Thr Ile Asn Asn Phe Asn TyrSer Asp Pro Val Asp Asn 1 5 10 15 AAA AAT ATT TTA TAT TTA GAT ACT CATTTA AAT ACA CTA GCT AAT GAG 96 Lys Asn Ile Leu Tyr Leu Asp Thr His LeuAsn Thr Leu Ala Asn Glu 20 25 30 CCT GAA AAA GCC TTT CGC ATT ACA GGA AATATA TGG GTA ATA CCT GAT 144 Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn IleTrp Val Ile Pro Asp 35 40 45 AGA TTT TCA AGA AAT TCT AAT CCA AAT TTA AATAAA CCT CCT CGA GTT 192 Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn LysPro Pro Arg Val 50 55 60 ACA AGC CCT AAA AGT GGT TAT TAT GAT CCT AAT TATTTG AGT ACT GAT 240 Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr LeuSer Thr Asp 65 70 75 80 TCT GAC AAA GAT ACA TTT TTA AAA GAA ATT ATA AAGTTA TTT AAA AGA 288 Ser Asp Lys Asp Thr Phe Leu Lys Glu Ile Ile Lys LeuPhe Lys Arg 85 90 95 ATT AAT TCT AGA GAA ATA GGA GAA GAA TTA ATA TAT AGACTT TCG ACA 336 Ile Asn Ser Arg Glu Ile Gly Glu Glu Leu Ile Tyr Arg LeuSer Thr 100 105 110 GAT ATA CCC TTT CCT GGG AAT AAC AAT ACT CCA ATT AATACT TTT GAT 384 Asp Ile Pro Phe Pro Gly Asn Asn Asn Thr Pro Ile Asn ThrPhe Asp 115 120 125 TTT GAT GTA GAT TTT AAC AGT GTT GAT GTT AAA ACT AGACAA GGT AAC 432 Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr Arg GlnGly Asn 130 135 140 AAC TGG GTT AAA ACT GGT AGC ATA AAT CCT AGT GTT ATAATA ACT GGA 480 Asn Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val Ile IleThr Gly 145 150 155 160 CCT AGA GAA AAC ATT ATA GAT CCA GAA ACT TCT ACGTTT AAA TTA ACT 528 Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr PheLys Leu Thr 165 170 175 AAC AAT ACT TTT GCG GCA CAA GAA GGA TTT GGT GCTTTA TCA ATA ATT 576 Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala LeuSer Ile Ile 180 185 190 TCA ATA TCA CCT AGA TTT ATG CTA ACA TAT AGT AATGCA ACT AAT GAT 624 Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn AlaThr Asn Asp 195 200 205 GTA GGA GAG GGT AGA TTT TCT AAG TCT GAA TTT TGCATG GAT CCA ATA 672 Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys MetAsp Pro Ile 210 215 220 CTA ATT TTA ATG CAT GAA CTT AAT CAT GCA ATG CATAAT TTA TAT GGA 720 Leu Ile Leu Met His Glu Leu Asn His Ala Met His AsnLeu Tyr Gly 225 230 235 240 ATA GCT ATA CCA AAT GAT CAA ACA ATT TCA TCTGTA ACT AGT AAT ATT 768 Ile Ala Ile Pro Asn Asp Gln Thr Ile Ser Ser ValThr Ser Asn Ile 245 250 255 TTT TAT TCT CAA TAT AAT GTG AAA TTA GAG TATGCA GAA ATA TAT GCA 816 Phe Tyr Ser Gln Tyr Asn Val Lys Leu Glu Tyr AlaGlu Ile Tyr Ala 260 265 270 TTT GGA GGT CCA ACT ATA GAC CTT ATT CCT AAAAGT GCA AGG AAA TAT 864 Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys SerAla Arg Lys Tyr 275 280 285 TTT GAG GAA AAG GCA TTG GAT TAT TAT AGA TCTATA GCT AAA AGA CTT 912 Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser IleAla Lys Arg Leu 290 295 300 AAT AGT ATA ACT ACT GCA AAT CCT TCA AGC TTTAAT AAA TAT ATA GGG 960 Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe AsnLys Tyr Ile Gly 305 310 315 320 GAA TAT AAA CAG AAA CTT ATT AGA AAG TATAGA TTC GTA GTA GAA TCT 1008 Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr ArgPhe Val Val Glu Ser 325 330 335 TCA GGT GAA GTT ACA GTA AAT CGT AAT AAGTTT GTT GAG TTA TAT AAT 1056 Ser Gly Glu Val Thr Val Asn Arg Asn Lys PheVal Glu Leu Tyr Asn 340 345 350 GAA CTT ACA CAA ATA TTT ACA GAA TTT AACTAC GCT AAA ATA TAT AAT 1104 Glu Leu Thr Gln Ile Phe Thr Glu Phe Asn TyrAla Lys Ile Tyr Asn 355 360 365 GTA CAA AAT AGG AAA ATA TAT CTT TCA AATGTA TAT ACT CCG GTT ACG 1152 Val Gln Asn Arg Lys Ile Tyr Leu Ser Asn ValTyr Thr Pro Val Thr 370 375 380 GCG AAT ATA TTA GAC GAT AAT GTT TAT GATATA CAA AAT GGA TTT AAT 1200 Ala Asn Ile Leu Asp Asp Asn Val Tyr Asp IleGln Asn Gly Phe Asn 385 390 395 400 ATA CCT AAA AGT AAT TTA AAT GTA CTATTT ATG GGT CAA AAT TTA TCT 1248 Ile Pro Lys Ser Asn Leu Asn Val Leu PheMet Gly Gln Asn Leu Ser 405 410 415 CGA AAT CCA GCA TTA AGA AAA GTC AATCCT GAA AAT ATG CTT TAT TTA 1296 Arg Asn Pro Ala Leu Arg Lys Val Asn ProGlu Asn Met Leu Tyr Leu 420 425 430 TTT ACA AAA TTT TGT CAT AAA GCA ATAGAT GGT AGA TCA TTA TAT AAT 1344 Phe Thr Lys Phe Cys His Lys Ala Ile AspGly Arg Ser Leu Tyr Asn 435 440 445 AAA ACA TTA GAT TGT AGA GAG CTT TTAGTT AAA AAT ACT GAC TTA CCC 1392 Lys Thr Leu Asp Cys Arg Glu Leu Leu ValLys Asn Thr Asp Leu Pro 450 455 460 TTT ATA GGT GAT ATT AGT GAT GTT AAAACT GAT ATA TTT TTA AGA AAA 1440 Phe Ile Gly Asp Ile Ser Asp Val Lys ThrAsp Ile Phe Leu Arg Lys 465 470 475 480 GAT ATT AAT GAA GAA ACT GAA GTTATA TAC TAT CCG GAC AAT GTT TCA 1488 Asp Ile Asn Glu Glu Thr Glu Val IleTyr Tyr Pro Asp Asn Val Ser 485 490 495 GTA GAT CAA GTT ATT CTC AGT AAGAAT ACC TCA GAA CAT GGA CAA CTA 1536 Val Asp Gln Val Ile Leu Ser Lys AsnThr Ser Glu His Gly Gln Leu 500 505 510 GAT TTA TTA TAC CCT AGT ATT GACAGT GAG AGT GAA ATA TTA CCA GGG 1584 Asp Leu Leu Tyr Pro Ser Ile Asp SerGlu Ser Glu Ile Leu Pro Gly 515 520 525 GAG AAT CAA GTC TTT TAT GAT AATAGA ACT CAA AAT GTT GAT TAT TTG 1632 Glu Asn Gln Val Phe Tyr Asp Asn ArgThr Gln Asn Val Asp Tyr Leu 530 535 540 AAT TCT TAT TAT TAC CTA GAA TCTCAA AAA CTA AGT GAT AAT GTT GAA 1680 Asn Ser Tyr Tyr Tyr Leu Glu Ser GlnLys Leu Ser Asp Asn Val Glu 545 550 555 560 GAT TTT ACT TTT ACG AGA TCAATT GAG GAG GCT TTG GAT AAT AGT GCA 1728 Asp Phe Thr Phe Thr Arg Ser IleGlu Glu Ala Leu Asp Asn Ser Ala 565 570 575 AAA GTA TAT ACT TAC TTT CCTACA CTA GCT AAT AAA GTA AAT GCG GGT 1776 Lys Val Tyr Thr Tyr Phe Pro ThrLeu Ala Asn Lys Val Asn Ala Gly 580 585 590 GTT CAA GGT GGT TTA TTT TTAATG TGG GCA AAT GAT GTA GTT GAA GAT 1824 Val Gln Gly Gly Leu Phe Leu MetTrp Ala Asn Asp Val Val Glu Asp 595 600 605 TTT ACT ACA AAT ATT CTA AGAAAA GAT ACA TTA GAT AAA ATA TCA GAT 1872 Phe Thr Thr Asn Ile Leu Arg LysAsp Thr Leu Asp Lys Ile Ser Asp 610 615 620 GTA TCA GCT ATT ATT CCC TATATA GGA CCC GCA TTA AAT ATA AGT AAT 1920 Val Ser Ala Ile Ile Pro Tyr IleGly Pro Ala Leu Asn Ile Ser Asn 625 630 635 640 TCT GTA AGA AGA GGA AATTTT ACT GAA GCA TTT GCA GTT ACT GGT GTA 1968 Ser Val Arg Arg Gly Asn PheThr Glu Ala Phe Ala Val Thr Gly Val 645 650 655 ACT ATT TTA TTA GAA GCATTT CCT GAA TTT ACA ATA CCT GCA CTT GGT 2016 Thr Ile Leu Leu Glu Ala PhePro Glu Phe Thr Ile Pro Ala Leu Gly 660 665 670 GCA TTT GTG ATT TAT AGTAAG GTT CAA GAA AGA AAC GAG ATT ATT AAA 2064 Ala Phe Val Ile Tyr Ser LysVal Gln Glu Arg Asn Glu Ile Ile Lys 675 680 685 ACT ATA GAT AAT TGT TTAGAA CAA AGG ATT AAG AGA TGG AAA GAT TCA 2112 Thr Ile Asp Asn Cys Leu GluGln Arg Ile Lys Arg Trp Lys Asp Ser 690 695 700 TAT GAA TGG ATG ATG GGAACG TGG TTA TCC AGG ATT ATT ACT CAA TTT 2160 Tyr Glu Trp Met Met Gly ThrTrp Leu Ser Arg Ile Ile Thr Gln Phe 705 710 715 720 AAT AAT ATA AGT TATCAA ATG TAT GAT TCT TTA AAT TAT CAG GCA GGT 2208 Asn Asn Ile Ser Tyr GlnMet Tyr Asp Ser Leu Asn Tyr Gln Ala Gly 725 730 735 GCA ATC AAA GCT AAAATA GAT TTA GAA TAT AAA AAA TAT TCA GGA AGT 2256 Ala Ile Lys Ala Lys IleAsp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser 740 745 750 GAT AAA GAA AAT ATAAAA AGT CAA GTT GAA AAT TTA AAA AAT AGT TTA 2304 Asp Lys Glu Asn Ile LysSer Gln Val Glu Asn Leu Lys Asn Ser Leu 755 760 765 GAT GTA AAA ATT TCGGAA GCA ATG AAT AAT ATA AAT AAA TTT ATA CGA 2352 Asp Val Lys Ile Ser GluAla Met Asn Asn Ile Asn Lys Phe Ile Arg 770 775 780 GAA TGT TCC GTA ACATAT TTA TTT AAA AAT ATG TTA CCT AAA GTA ATT 2400 Glu Cys Ser Val Thr TyrLeu Phe Lys Asn Met Leu Pro Lys Val Ile 785 790 795 800 GAT GAA TTA AATGAG TTT GAT CGA AAT ACT AAA GCA AAA TTA ATT AAT 2448 Asp Glu Leu Asn GluPhe Asp Arg Asn Thr Lys Ala Lys Leu Ile Asn 805 810 815 CTT ATA GAT AGTCAT AAT ATT ATT CTA GTT GGT GAA GTA GAT AAA TTA 2496 Leu Ile Asp Ser HisAsn Ile Ile Leu Val Gly Glu Val Asp Lys Leu 820 825 830 AAA GCA AAA GTAAAT AAT AGC TTT CAA AAT ACA ATA CCC TTT AAT ATT 2544 Lys Ala Lys Val AsnAsn Ser Phe Gln Asn Thr Ile Pro Phe Asn Ile 835 840 845 TTT TCA TAT ACTAAT AAT TCT TTA TTA AAA GAT ATA ATT AAT GAA TAT 2592 Phe Ser Tyr Thr AsnAsn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr 850 855 860 TTC AAT AAT ATTAAT GAT TCA AAA ATT TTG AGC CTA CAA AAC AGA AAA 2640 Phe Asn Asn Ile AsnAsp Ser Lys Ile Leu Ser Leu Gln Asn Arg Lys 865 870 875 880 AAT ACT TTAGTG GAT ACA TCA GGA TAT AAT GCA GAA GTG AGT GAA GAA 2688 Asn Thr Leu ValAsp Thr Ser Gly Tyr Asn Ala Glu Val Ser Glu Glu 885 890 895 GGC GAT GTTCAG CTT AAT CCA ATA TTT CCA TTT GAC TTT AAA TTA GGT 2736 Gly Asp Val GlnLeu Asn Pro Ile Phe Pro Phe Asp Phe Lys Leu Gly 900 905 910 AGT TCA GGGGAG GAT AGA GGT AAA GTT ATA GTA ACC CAG AAT GAA AAT 2784 Ser Ser Gly GluAsp Arg Gly Lys Val Ile Val Thr Gln Asn Glu Asn 915 920 925 ATT GTA TATAAT TCT ATG TAT GAA AGT TTT AGC ATT AGT TTT TGG ATT 2832 Ile Val Tyr AsnSer Met Tyr Glu Ser Phe Ser Ile Ser Phe Trp Ile 930 935 940 AGA ATA AATAAA TGG GTA AGT AAT TTA CCT GGA TAT ACT ATA ATT GAT 2880 Arg Ile Asn LysTrp Val Ser Asn Leu Pro Gly Tyr Thr Ile Ile Asp 945 950 955 960 AGT GTTAAA AAT AAC TCA GGT TGG AGT ATA GGT ATT ATT AGT AAT TTT 2928 Ser Val LysAsn Asn Ser Gly Trp Ser Ile Gly Ile Ile Ser Asn Phe 965 970 975 TTA GTATTT ACT TTA AAA CAA AAT GAA GAT AGT GAA CAA AGT ATA AAT 2976 Leu Val PheThr Leu Lys Gln Asn Glu Asp Ser Glu Gln Ser Ile Asn 980 985 990 TTT AGTTAT GAT ATA TCA AAT AAT GCT CCT GGA TAC AAT AAA TGG TTT 3024 Phe Ser TyrAsp Ile Ser Asn Asn Ala Pro Gly Tyr Asn Lys Trp Phe 995 1000 1005 TTTGTA ACT GTT ACT AAC AAT ATG ATG GGA AAT ATG AAG ATT TAT ATA 3072 Phe ValThr Val Thr Asn Asn Met Met Gly Asn Met Lys Ile Tyr Ile 1010 1015 1020AAT GGA AAA TTA ATA GAT ACT ATA AAA GTT AAA GAA CTA ACT GGA ATT 3120 AsnGly Lys Leu Ile Asp Thr Ile Lys Val Lys Glu Leu Thr Gly Ile 1025 10301035 1040 AAT TTT AGC AAA ACT ATA ACA TTT GAA ATA AAT AAA ATT CCA GATACC 3168 Asn Phe Ser Lys Thr Ile Thr Phe Glu Ile Asn Lys Ile Pro Asp Thr1045 1050 1055 GGT TTG ATT ACT TCA GAT TCT GAT AAC ATC AAT ATG TGG ATAAGA GAT 3216 Gly Leu Ile Thr Ser Asp Ser Asp Asn Ile Asn Met Trp Ile ArgAsp 1060 1065 1070 TTT TAT ATA TTT GCT AAA GAA TTA GAT GGT AAA GAT ATTAAT ATA TTA 3264 Phe Tyr Ile Phe Ala Lys Glu Leu Asp Gly Lys Asp Ile AsnIle Leu 1075 1080 1085 TTT AAT AGC TTG CAA TAT ACT AAT GTT GTA AAA GATTAT TGG GGA AAT 3312 Phe Asn Ser Leu Gln Tyr Thr Asn Val Val Lys Asp TyrTrp Gly Asn 1090 1095 1100 GAT TTA AGA TAT AAT AAA GAA TAT TAT ATG GTTAAT ATA GAT TAT TTA 3360 Asp Leu Arg Tyr Asn Lys Glu Tyr Tyr Met Val AsnIle Asp Tyr Leu 1105 1110 1115 1120 AAT AGA TAT ATG TAT GCG AAC TCA CGACAA ATT GTT TTT AAT ACA CGT 3408 Asn Arg Tyr Met Tyr Ala Asn Ser Arg GlnIle Val Phe Asn Thr Arg 1125 1130 1135 AGA AAT AAT AAT GAC TTC AAT GAAGGA TAT AAA ATT ATA ATA AAA AGA 3456 Arg Asn Asn Asn Asp Phe Asn Glu GlyTyr Lys Ile Ile Ile Lys Arg 1140 1145 1150 ATC AGA GGA AAT ACA AAT GATACT AGA GTA CGA GGA GGA GAT ATT TTA 3504 Ile Arg Gly Asn Thr Asn Asp ThrArg Val Arg Gly Gly Asp Ile Leu 1155 1160 1165 TAT TTT GAT ATG ACA ATTAAT AAC AAA GCA TAT AAT TTG TTT ATG AAG 3552 Tyr Phe Asp Met Thr Ile AsnAsn Lys Ala Tyr Asn Leu Phe Met Lys 1170 1175 1180 AAT GAA ACT ATG TATGCA GAT AAT CAT AGT ACT GAA GAT ATA TAT GCT 3600 Asn Glu Thr Met Tyr AlaAsp Asn His Ser Thr Glu Asp Ile Tyr Ala 1185 1190 1195 1200 ATA GGT TTAAGA GAA CAA ACA AAG GAT ATA AAT GAT AAT ATT ATA TTT 3648 Ile Gly Leu ArgGlu Gln Thr Lys Asp Ile Asn Asp Asn Ile Ile Phe 1205 1210 1215 CAA ATACAA CCA ATG AAT AAT ACT TAT TAT TAC GCA TCT CAA ATA TTT 3696 Gln Ile GlnPro Met Asn Asn Thr Tyr Tyr Tyr Ala Ser Gln Ile Phe 1220 1225 1230 AAATCA AAT TTT AAT GGA GAA AAT ATT TCT GGA ATA TGT TCA ATA GGT 3744 Lys SerAsn Phe Asn Gly Glu Asn Ile Ser Gly Ile Cys Ser Ile Gly 1235 1240 1245ACT TAT CGT TTT AGA CTT GGA GGT GAT TGG TAT AGA CAC AAT TAT TTG 3792 ThrTyr Arg Phe Arg Leu Gly Gly Asp Trp Tyr Arg His Asn Tyr Leu 1250 12551260 GTG CCT ACT GTG AAG CAA GGA AAT TAT GCT TCA TTA TTA GAA TCA ACA3840 Val Pro Thr Val Lys Gln Gly Asn Tyr Ala Ser Leu Leu Glu Ser Thr1265 1270 1275 1280 TCA ACT CAT TGG GGT TTT GTA CCT GTA AGT GAA TAA 3876Ser Thr His Trp Gly Phe Val Pro Val Ser Glu 1285 1290 1291 amino acidsamino acid linear protein 60 Met Pro Ile Thr Ile Asn Asn Phe Asn Tyr SerAsp Pro Val Asp Asn 1 5 10 15 Lys Asn Ile Leu Tyr Leu Asp Thr His LeuAsn Thr Leu Ala Asn Glu 20 25 30 Pro Glu Lys Ala Phe Arg Ile Thr Gly AsnIle Trp Val Ile Pro Asp 35 40 45 Arg Phe Ser Arg Asn Ser Asn Pro Asn LeuAsn Lys Pro Pro Arg Val 50 55 60 Thr Ser Pro Lys Ser Gly Tyr Tyr Asp ProAsn Tyr Leu Ser Thr Asp 65 70 75 80 Ser Asp Lys Asp Thr Phe Leu Lys GluIle Ile Lys Leu Phe Lys Arg 85 90 95 Ile Asn Ser Arg Glu Ile Gly Glu GluLeu Ile Tyr Arg Leu Ser Thr 100 105 110 Asp Ile Pro Phe Pro Gly Asn AsnAsn Thr Pro Ile Asn Thr Phe Asp 115 120 125 Phe Asp Val Asp Phe Asn SerVal Asp Val Lys Thr Arg Gln Gly Asn 130 135 140 Asn Trp Val Lys Thr GlySer Ile Asn Pro Ser Val Ile Ile Thr Gly 145 150 155 160 Pro Arg Glu AsnIle Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr 165 170 175 Asn Asn ThrPhe Ala Ala Gln Glu Gly Phe Gly Ala Leu Ser Ile Ile 180 185 190 Ser IleSer Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr Asn Asp 195 200 205 ValGly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met Asp Pro Ile 210 215 220Leu Ile Leu Met His Glu Leu Asn His Ala Met His Asn Leu Tyr Gly 225 230235 240 Ile Ala Ile Pro Asn Asp Gln Thr Ile Ser Ser Val Thr Ser Asn Ile245 250 255 Phe Tyr Ser Gln Tyr Asn Val Lys Leu Glu Tyr Ala Glu Ile TyrAla 260 265 270 Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser Ala ArgLys Tyr 275 280 285 Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile AlaLys Arg Leu 290 295 300 Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe AsnLys Tyr Ile Gly 305 310 315 320 Glu Tyr Lys Gln Lys Leu Ile Arg Lys TyrArg Phe Val Val Glu Ser 325 330 335 Ser Gly Glu Val Thr Val Asn Arg AsnLys Phe Val Glu Leu Tyr Asn 340 345 350 Glu Leu Thr Gln Ile Phe Thr GluPhe Asn Tyr Ala Lys Ile Tyr Asn 355 360 365 Val Gln Asn Arg Lys Ile TyrLeu Ser Asn Val Tyr Thr Pro Val Thr 370 375 380 Ala Asn Ile Leu Asp AspAsn Val Tyr Asp Ile Gln Asn Gly Phe Asn 385 390 395 400 Ile Pro Lys SerAsn Leu Asn Val Leu Phe Met Gly Gln Asn Leu Ser 405 410 415 Arg Asn ProAla Leu Arg Lys Val Asn Pro Glu Asn Met Leu Tyr Leu 420 425 430 Phe ThrLys Phe Cys His Lys Ala Ile Asp Gly Arg Ser Leu Tyr Asn 435 440 445 LysThr Leu Asp Cys Arg Glu Leu Leu Val Lys Asn Thr Asp Leu Pro 450 455 460Phe Ile Gly Asp Ile Ser Asp Val Lys Thr Asp Ile Phe Leu Arg Lys 465 470475 480 Asp Ile Asn Glu Glu Thr Glu Val Ile Tyr Tyr Pro Asp Asn Val Ser485 490 495 Val Asp Gln Val Ile Leu Ser Lys Asn Thr Ser Glu His Gly GlnLeu 500 505 510 Asp Leu Leu Tyr Pro Ser Ile Asp Ser Glu Ser Glu Ile LeuPro Gly 515 520 525 Glu Asn Gln Val Phe Tyr Asp Asn Arg Thr Gln Asn ValAsp Tyr Leu 530 535 540 Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu SerAsp Asn Val Glu 545 550 555 560 Asp Phe Thr Phe Thr Arg Ser Ile Glu GluAla Leu Asp Asn Ser Ala 565 570 575 Lys Val Tyr Thr Tyr Phe Pro Thr LeuAla Asn Lys Val Asn Ala Gly 580 585 590 Val Gln Gly Gly Leu Phe Leu MetTrp Ala Asn Asp Val Val Glu Asp 595 600 605 Phe Thr Thr Asn Ile Leu ArgLys Asp Thr Leu Asp Lys Ile Ser Asp 610 615 620 Val Ser Ala Ile Ile ProTyr Ile Gly Pro Ala Leu Asn Ile Ser Asn 625 630 635 640 Ser Val Arg ArgGly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val 645 650 655 Thr Ile LeuLeu Glu Ala Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly 660 665 670 Ala PheVal Ile Tyr Ser Lys Val Gln Glu Arg Asn Glu Ile Ile Lys 675 680 685 ThrIle Asp Asn Cys Leu Glu Gln Arg Ile Lys Arg Trp Lys Asp Ser 690 695 700Tyr Glu Trp Met Met Gly Thr Trp Leu Ser Arg Ile Ile Thr Gln Phe 705 710715 720 Asn Asn Ile Ser Tyr Gln Met Tyr Asp Ser Leu Asn Tyr Gln Ala Gly725 730 735 Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser GlySer 740 745 750 Asp Lys Glu Asn Ile Lys Ser Gln Val Glu Asn Leu Lys AsnSer Leu 755 760 765 Asp Val Lys Ile Ser Glu Ala Met Asn Asn Ile Asn LysPhe Ile Arg 770 775 780 Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met LeuPro Lys Val Ile 785 790 795 800 Asp Glu Leu Asn Glu Phe Asp Arg Asn ThrLys Ala Lys Leu Ile Asn 805 810 815 Leu Ile Asp Ser His Asn Ile Ile LeuVal Gly Glu Val Asp Lys Leu 820 825 830 Lys Ala Lys Val Asn Asn Ser PheGln Asn Thr Ile Pro Phe Asn Ile 835 840 845 Phe Ser Tyr Thr Asn Asn SerLeu Leu Lys Asp Ile Ile Asn Glu Tyr 850 855 860 Phe Asn Asn Ile Asn AspSer Lys Ile Leu Ser Leu Gln Asn Arg Lys 865 870 875 880 Asn Thr Leu ValAsp Thr Ser Gly Tyr Asn Ala Glu Val Ser Glu Glu 885 890 895 Gly Asp ValGln Leu Asn Pro Ile Phe Pro Phe Asp Phe Lys Leu Gly 900 905 910 Ser SerGly Glu Asp Arg Gly Lys Val Ile Val Thr Gln Asn Glu Asn 915 920 925 IleVal Tyr Asn Ser Met Tyr Glu Ser Phe Ser Ile Ser Phe Trp Ile 930 935 940Arg Ile Asn Lys Trp Val Ser Asn Leu Pro Gly Tyr Thr Ile Ile Asp 945 950955 960 Ser Val Lys Asn Asn Ser Gly Trp Ser Ile Gly Ile Ile Ser Asn Phe965 970 975 Leu Val Phe Thr Leu Lys Gln Asn Glu Asp Ser Glu Gln Ser IleAsn 980 985 990 Phe Ser Tyr Asp Ile Ser Asn Asn Ala Pro Gly Tyr Asn LysTrp Phe 995 1000 1005 Phe Val Thr Val Thr Asn Asn Met Met Gly Asn MetLys Ile Tyr Ile 1010 1015 1020 Asn Gly Lys Leu Ile Asp Thr Ile Lys ValLys Glu Leu Thr Gly Ile 1025 1030 1035 1040 Asn Phe Ser Lys Thr Ile ThrPhe Glu Ile Asn Lys Ile Pro Asp Thr 1045 1050 1055 Gly Leu Ile Thr SerAsp Ser Asp Asn Ile Asn Met Trp Ile Arg Asp 1060 1065 1070 Phe Tyr IlePhe Ala Lys Glu Leu Asp Gly Lys Asp Ile Asn Ile Leu 1075 1080 1085 PheAsn Ser Leu Gln Tyr Thr Asn Val Val Lys Asp Tyr Trp Gly Asn 1090 10951100 Asp Leu Arg Tyr Asn Lys Glu Tyr Tyr Met Val Asn Ile Asp Tyr Leu1105 1110 1115 1120 Asn Arg Tyr Met Tyr Ala Asn Ser Arg Gln Ile Val PheAsn Thr Arg 1125 1130 1135 Arg Asn Asn Asn Asp Phe Asn Glu Gly Tyr LysIle Ile Ile Lys Arg 1140 1145 1150 Ile Arg Gly Asn Thr Asn Asp Thr ArgVal Arg Gly Gly Asp Ile Leu 1155 1160 1165 Tyr Phe Asp Met Thr Ile AsnAsn Lys Ala Tyr Asn Leu Phe Met Lys 1170 1175 1180 Asn Glu Thr Met TyrAla Asp Asn His Ser Thr Glu Asp Ile Tyr Ala 1185 1190 1195 1200 Ile GlyLeu Arg Glu Gln Thr Lys Asp Ile Asn Asp Asn Ile Ile Phe 1205 1210 1215Gln Ile Gln Pro Met Asn Asn Thr Tyr Tyr Tyr Ala Ser Gln Ile Phe 12201225 1230 Lys Ser Asn Phe Asn Gly Glu Asn Ile Ser Gly Ile Cys Ser IleGly 1235 1240 1245 Thr Tyr Arg Phe Arg Leu Gly Gly Asp Trp Tyr Arg HisAsn Tyr Leu 1250 1255 1260 Val Pro Thr Val Lys Gln Gly Asn Tyr Ala SerLeu Leu Glu Ser Thr 1265 1270 1275 1280 Ser Thr His Trp Gly Phe Val ProVal Ser Glu 1285 1290 1502 base pairs nucleic acid double linear DNA(genomic) CDS 108..1493 61 AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGGGAATTGTGAG CGGATAACAA 60 TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAGATATACC ATG GGC CAT 116 Met Gly His 1 CAT CAT CAT CAT CAT CAT CAT CATCAC AGC AGC GGC CAT ATC GAA GGT 164 His His His His His His His His HisSer Ser Gly His Ile Glu Gly 5 10 15 CGT CAT ATG GCT AGC ATG GCT TTA TTAAAA GAT ATA ATT AAT GAA TAT 212 Arg His Met Ala Ser Met Ala Leu Leu LysAsp Ile Ile Asn Glu Tyr 20 25 30 35 TTC AAT AAT ATT AAT GAT TCA AAA ATTTTG AGC CTA CAA AAC AGA AAA 260 Phe Asn Asn Ile Asn Asp Ser Lys Ile LeuSer Leu Gln Asn Arg Lys 40 45 50 AAT ACT TTA GTG GAT ACA TCA GGA TAT AATGCA GAA GTG AGT GAA GAA 308 Asn Thr Leu Val Asp Thr Ser Gly Tyr Asn AlaGlu Val Ser Glu Glu 55 60 65 GGC GAT GTT CAG CTT AAT CCA ATA TTT CCA TTTGAC TTT AAA TTA GGT 356 Gly Asp Val Gln Leu Asn Pro Ile Phe Pro Phe AspPhe Lys Leu Gly 70 75 80 AGT TCA GGG GAG GAT AGA GGT AAA GTT ATA GTA ACCCAG AAT GAA AAT 404 Ser Ser Gly Glu Asp Arg Gly Lys Val Ile Val Thr GlnAsn Glu Asn 85 90 95 ATT GTA TAT AAT TCT ATG TAT GAA AGT TTT AGC ATT AGTTTT TGG ATT 452 Ile Val Tyr Asn Ser Met Tyr Glu Ser Phe Ser Ile Ser PheTrp Ile 100 105 110 115 AGA ATA AAT AAA TGG GTA AGT AAT TTA CCT GGA TATACT ATA ATT GAT 500 Arg Ile Asn Lys Trp Val Ser Asn Leu Pro Gly Tyr ThrIle Ile Asp 120 125 130 AGT GTT AAA AAT AAC TCA GGT TGG AGT ATA GGT ATTATT AGT AAT TTT 548 Ser Val Lys Asn Asn Ser Gly Trp Ser Ile Gly Ile IleSer Asn Phe 135 140 145 TTA GTA TTT ACT TTA AAA CAA AAT GAA GAT AGT GAACAA AGT ATA AAT 596 Leu Val Phe Thr Leu Lys Gln Asn Glu Asp Ser Glu GlnSer Ile Asn 150 155 160 TTT AGT TAT GAT ATA TCA AAT AAT GCT CCT GGA TACAAT AAA TGG TTT 644 Phe Ser Tyr Asp Ile Ser Asn Asn Ala Pro Gly Tyr AsnLys Trp Phe 165 170 175 TTT GTA ACT GTT ACT AAC AAT ATG ATG GGA AAT ATGAAG ATT TAT ATA 692 Phe Val Thr Val Thr Asn Asn Met Met Gly Asn Met LysIle Tyr Ile 180 185 190 195 AAT GGA AAA TTA ATA GAT ACT ATA AAA GTT AAAGAA CTA ACT GGA ATT 740 Asn Gly Lys Leu Ile Asp Thr Ile Lys Val Lys GluLeu Thr Gly Ile 200 205 210 AAT TTT AGC AAA ACT ATA ACA TTT GAA ATA AATAAA ATT CCA GAT ACC 788 Asn Phe Ser Lys Thr Ile Thr Phe Glu Ile Asn LysIle Pro Asp Thr 215 220 225 GGT TTG ATT ACT TCA GAT TCT GAT AAC ATC AATATG TGG ATA AGA GAT 836 Gly Leu Ile Thr Ser Asp Ser Asp Asn Ile Asn MetTrp Ile Arg Asp 230 235 240 TTT TAT ATA TTT GCT AAA GAA TTA GAT GGT AAAGAT ATT AAT ATA TTA 884 Phe Tyr Ile Phe Ala Lys Glu Leu Asp Gly Lys AspIle Asn Ile Leu 245 250 255 TTT AAT AGC TTG CAA TAT ACT AAT GTT GTA AAAGAT TAT TGG GGA AAT 932 Phe Asn Ser Leu Gln Tyr Thr Asn Val Val Lys AspTyr Trp Gly Asn 260 265 270 275 GAT TTA AGA TAT AAT AAA GAA TAT TAT ATGGTT AAT ATA GAT TAT TTA 980 Asp Leu Arg Tyr Asn Lys Glu Tyr Tyr Met ValAsn Ile Asp Tyr Leu 280 285 290 AAT AGA TAT ATG TAT GCG AAC TCA CGA CAAATT GTT TTT AAT ACA CGT 1028 Asn Arg Tyr Met Tyr Ala Asn Ser Arg Gln IleVal Phe Asn Thr Arg 295 300 305 AGA AAT AAT AAT GAC TTC AAT GAA GGA TATAAA ATT ATA ATA AAA AGA 1076 Arg Asn Asn Asn Asp Phe Asn Glu Gly Tyr LysIle Ile Ile Lys Arg 310 315 320 ATC AGA GGA AAT ACA AAT GAT ACT AGA GTACGA GGA GGA GAT ATT TTA 1124 Ile Arg Gly Asn Thr Asn Asp Thr Arg Val ArgGly Gly Asp Ile Leu 325 330 335 TAT TTT GAT ATG ACA ATT AAT AAC AAA GCATAT AAT TTG TTT ATG AAG 1172 Tyr Phe Asp Met Thr Ile Asn Asn Lys Ala TyrAsn Leu Phe Met Lys 340 345 350 355 AAT GAA ACT ATG TAT GCA GAT AAT CATAGT ACT GAA GAT ATA TAT GCT 1220 Asn Glu Thr Met Tyr Ala Asp Asn His SerThr Glu Asp Ile Tyr Ala 360 365 370 ATA GGT TTA AGA GAA CAA ACA AAG GATATA AAT GAT AAT ATT ATA TTT 1268 Ile Gly Leu Arg Glu Gln Thr Lys Asp IleAsn Asp Asn Ile Ile Phe 375 380 385 CAA ATA CAA CCA ATG AAT AAT ACT TATTAT TAC GCA TCT CAA ATA TTT 1316 Gln Ile Gln Pro Met Asn Asn Thr Tyr TyrTyr Ala Ser Gln Ile Phe 390 395 400 AAA TCA AAT TTT AAT GGA GAA AAT ATTTCT GGA ATA TGT TCA ATA GGT 1364 Lys Ser Asn Phe Asn Gly Glu Asn Ile SerGly Ile Cys Ser Ile Gly 405 410 415 ACT TAT CGT TTT AGA CTT GGA GGT GATTGG TAT AGA CAC AAT TAT TTG 1412 Thr Tyr Arg Phe Arg Leu Gly Gly Asp TrpTyr Arg His Asn Tyr Leu 420 425 430 435 GTG CCT ACT GTG AAG CAA GGA AATTAT GCT TCA TTA TTA GAA TCA ACA 1460 Val Pro Thr Val Lys Gln Gly Asn TyrAla Ser Leu Leu Glu Ser Thr 440 445 450 TCA ACT CAT TGG GGT TTT GTA CCTGTA AGT GAA TAAAAGCTT 1502 Ser Thr His Trp Gly Phe Val Pro Val Ser Glu455 460 462 amino acids amino acid linear protein 62 Met Gly His His HisHis His His His His His His Ser Ser Gly His 1 5 10 15 Ile Glu Gly ArgHis Met Ala Ser Met Ala Leu Leu Lys Asp Ile Ile 20 25 30 Asn Glu Tyr PheAsn Asn Ile Asn Asp Ser Lys Ile Leu Ser Leu Gln 35 40 45 Asn Arg Lys AsnThr Leu Val Asp Thr Ser Gly Tyr Asn Ala Glu Val 50 55 60 Ser Glu Glu GlyAsp Val Gln Leu Asn Pro Ile Phe Pro Phe Asp Phe 65 70 75 80 Lys Leu GlySer Ser Gly Glu Asp Arg Gly Lys Val Ile Val Thr Gln 85 90 95 Asn Glu AsnIle Val Tyr Asn Ser Met Tyr Glu Ser Phe Ser Ile Ser 100 105 110 Phe TrpIle Arg Ile Asn Lys Trp Val Ser Asn Leu Pro Gly Tyr Thr 115 120 125 IleIle Asp Ser Val Lys Asn Asn Ser Gly Trp Ser Ile Gly Ile Ile 130 135 140Ser Asn Phe Leu Val Phe Thr Leu Lys Gln Asn Glu Asp Ser Glu Gln 145 150155 160 Ser Ile Asn Phe Ser Tyr Asp Ile Ser Asn Asn Ala Pro Gly Tyr Asn165 170 175 Lys Trp Phe Phe Val Thr Val Thr Asn Asn Met Met Gly Asn MetLys 180 185 190 Ile Tyr Ile Asn Gly Lys Leu Ile Asp Thr Ile Lys Val LysGlu Leu 195 200 205 Thr Gly Ile Asn Phe Ser Lys Thr Ile Thr Phe Glu IleAsn Lys Ile 210 215 220 Pro Asp Thr Gly Leu Ile Thr Ser Asp Ser Asp AsnIle Asn Met Trp 225 230 235 240 Ile Arg Asp Phe Tyr Ile Phe Ala Lys GluLeu Asp Gly Lys Asp Ile 245 250 255 Asn Ile Leu Phe Asn Ser Leu Gln TyrThr Asn Val Val Lys Asp Tyr 260 265 270 Trp Gly Asn Asp Leu Arg Tyr AsnLys Glu Tyr Tyr Met Val Asn Ile 275 280 285 Asp Tyr Leu Asn Arg Tyr MetTyr Ala Asn Ser Arg Gln Ile Val Phe 290 295 300 Asn Thr Arg Arg Asn AsnAsn Asp Phe Asn Glu Gly Tyr Lys Ile Ile 305 310 315 320 Ile Lys Arg IleArg Gly Asn Thr Asn Asp Thr Arg Val Arg Gly Gly 325 330 335 Asp Ile LeuTyr Phe Asp Met Thr Ile Asn Asn Lys Ala Tyr Asn Leu 340 345 350 Phe MetLys Asn Glu Thr Met Tyr Ala Asp Asn His Ser Thr Glu Asp 355 360 365 IleTyr Ala Ile Gly Leu Arg Glu Gln Thr Lys Asp Ile Asn Asp Asn 370 375 380Ile Ile Phe Gln Ile Gln Pro Met Asn Asn Thr Tyr Tyr Tyr Ala Ser 385 390395 400 Gln Ile Phe Lys Ser Asn Phe Asn Gly Glu Asn Ile Ser Gly Ile Cys405 410 415 Ser Ile Gly Thr Tyr Arg Phe Arg Leu Gly Gly Asp Trp Tyr ArgHis 420 425 430 Asn Tyr Leu Val Pro Thr Val Lys Gln Gly Asn Tyr Ala SerLeu Leu 435 440 445 Glu Ser Thr Ser Thr His Trp Gly Phe Val Pro Val SerGlu 450 455 460 32 base pairs nucleic acid single linear other nucleicacid /desc = “DNA” 63 CGCCATGGCT TTATTAAAAG ATATAATTAA TG 32 32 basepairs nucleic acid single linear other nucleic acid /desc = “DNA” 64GCAAGCTTTT ATTCACTTAC AGGTACAAAA CC 32 3831 base pairs nucleic aciddouble linear DNA (genomic) CDS 1..3828 65 ATG ACA TGG CCA GTA AAA GATTTT AAT TAT AGT GAT CCT GTT AAT GAC 48 Met Thr Trp Pro Val Lys Asp PheAsn Tyr Ser Asp Pro Val Asn Asp 1 5 10 15 AAT GAT ATA TTA TAT TTA AGAATA CCA CAA AAT AAG TTA ATT ACT ACA 96 Asn Asp Ile Leu Tyr Leu Arg IlePro Gln Asn Lys Leu Ile Thr Thr 20 25 30 CCT GTA AAA GCT TTT ATG ATT ACTCAA AAT ATT TGG GTA ATA CCA GAA 144 Pro Val Lys Ala Phe Met Ile Thr GlnAsn Ile Trp Val Ile Pro Glu 35 40 45 AGA TTT TCA TCA GAT ACT AAT CCA AGTTTA AGT AAA CCG CCC AGA CCT 192 Arg Phe Ser Ser Asp Thr Asn Pro Ser LeuSer Lys Pro Pro Arg Pro 50 55 60 ACT TCA AAG TAT CAA AGT TAT TAT GAT CCTAGT TAT TTA TCT ACT GAT 240 Thr Ser Lys Tyr Gln Ser Tyr Tyr Asp Pro SerTyr Leu Ser Thr Asp 65 70 75 80 GAA CAA AAA GAT ACA TTT TTA AAA GGG ATTATA AAA TTA TTT AAA AGA 288 Glu Gln Lys Asp Thr Phe Leu Lys Gly Ile IleLys Leu Phe Lys Arg 85 90 95 ATT AAT GAA AGA GAT ATA GGA AAA AAA TTA ATAAAT TAT TTA GTA GTT 336 Ile Asn Glu Arg Asp Ile Gly Lys Lys Leu Ile AsnTyr Leu Val Val 100 105 110 GGT TCA CCT TTT ATG GGA GAT TCA AGT ACG CCTGAA GAT ACA TTT GAT 384 Gly Ser Pro Phe Met Gly Asp Ser Ser Thr Pro GluAsp Thr Phe Asp 115 120 125 TTT ACA CGT CAT ACT ACT AAT ATT GCA GTT GAAAAG TTT GAA AAT GGT 432 Phe Thr Arg His Thr Thr Asn Ile Ala Val Glu LysPhe Glu Asn Gly 130 135 140 AGT TGG AAA GTA ACA AAT ATT ATA ACA CCA AGTGTA TTG ATA TTT GGA 480 Ser Trp Lys Val Thr Asn Ile Ile Thr Pro Ser ValLeu Ile Phe Gly 145 150 155 160 CCA CTT CCT AAT ATA TTA GAC TAT ACA GCATCC CTT ACA TTG CAA GGA 528 Pro Leu Pro Asn Ile Leu Asp Tyr Thr Ala SerLeu Thr Leu Gln Gly 165 170 175 CAA CAA TCA AAT CCA TCA TTT GAA GGG TTTGGA ACA TTA TCT ATA CTA 576 Gln Gln Ser Asn Pro Ser Phe Glu Gly Phe GlyThr Leu Ser Ile Leu 180 185 190 AAA GTA GCA CCT GAA TTT TTG TTA ACA TTTAGT GAT GTA ACA TCT AAT 624 Lys Val Ala Pro Glu Phe Leu Leu Thr Phe SerAsp Val Thr Ser Asn 195 200 205 CAA AGT TCA GCT GTA TTA GGC AAA TCT ATATTT TGT ATG GAT CCA GTA 672 Gln Ser Ser Ala Val Leu Gly Lys Ser Ile PheCys Met Asp Pro Val 210 215 220 ATA GCT TTA ATG CAT GAG TTA ACA CAT TCTTTG CAT CAA TTA TAT GGA 720 Ile Ala Leu Met His Glu Leu Thr His Ser LeuHis Gln Leu Tyr Gly 225 230 235 240 ATA AAT ATA CCA TCT GAT AAA AGG ATTCGT CCA CAA GTT AGC GAG GGA 768 Ile Asn Ile Pro Ser Asp Lys Arg Ile ArgPro Gln Val Ser Glu Gly 245 250 255 TTT TTC TCT CAA GAT GGA CCC AAC GTACAA TTT GAG GAA TTA TAT ACA 816 Phe Phe Ser Gln Asp Gly Pro Asn Val GlnPhe Glu Glu Leu Tyr Thr 260 265 270 TTT GGA GGA TTA GAT GTT GAA ATA ATACCT CAA ATT GAA AGA TCA CAA 864 Phe Gly Gly Leu Asp Val Glu Ile Ile ProGln Ile Glu Arg Ser Gln 275 280 285 TTA AGA GAA AAA GCA TTA GGT CAC TATAAA GAT ATA GCG AAA AGA CTT 912 Leu Arg Glu Lys Ala Leu Gly His Tyr LysAsp Ile Ala Lys Arg Leu 290 295 300 AAT AAT ATT AAT AAA ACT ATT CCT TCTAGT TGG ATT AGT AAT ATA GAT 960 Asn Asn Ile Asn Lys Thr Ile Pro Ser SerTrp Ile Ser Asn Ile Asp 305 310 315 320 AAA TAT AAA AAA ATA TTT TCT GAAAAG TAT AAT TTT GAT AAA GAT AAT 1008 Lys Tyr Lys Lys Ile Phe Ser Glu LysTyr Asn Phe Asp Lys Asp Asn 325 330 335 ACA GGA AAT TTT GTT GTA AAT ATTGAT AAA TTC AAT AGC TTA TAT TCA 1056 Thr Gly Asn Phe Val Val Asn Ile AspLys Phe Asn Ser Leu Tyr Ser 340 345 350 GAC TTG ACT AAT GTT ATG TCA GAAGTT GTT TAT TCT TCG CAA TAT AAT 1104 Asp Leu Thr Asn Val Met Ser Glu ValVal Tyr Ser Ser Gln Tyr Asn 355 360 365 GTT AAA AAC AGG ACT CAT TAT TTTTCA AGG CAT TAT CTA CCT GTA TTT 1152 Val Lys Asn Arg Thr His Tyr Phe SerArg His Tyr Leu Pro Val Phe 370 375 380 GCA AAT ATA TTA GAT GAT AAT ATTTAT ACT ATA AGA GAT GGT TTT AAT 1200 Ala Asn Ile Leu Asp Asp Asn Ile TyrThr Ile Arg Asp Gly Phe Asn 385 390 395 400 TTA ACA AAT AAA GGT TTT AATATA GAA AAT TCG GGT CAG AAT ATA GAA 1248 Leu Thr Asn Lys Gly Phe Asn IleGlu Asn Ser Gly Gln Asn Ile Glu 405 410 415 AGG AAT CCT GCA CTA CAA AAGCTT AGT TCA GAA AGT GTA GTA GAT TTA 1296 Arg Asn Pro Ala Leu Gln Lys LeuSer Ser Glu Ser Val Val Asp Leu 420 425 430 TTT ACA AAA GTA TGT TTA AGATTA ACA AAA AAT AGT AGA GAT GAT TCA 1344 Phe Thr Lys Val Cys Leu Arg LeuThr Lys Asn Ser Arg Asp Asp Ser 435 440 445 ACA TGT ATT AAA GTT AAA AATAAT AGA TTA CCT TAT GTA GCT GAT AAA 1392 Thr Cys Ile Lys Val Lys Asn AsnArg Leu Pro Tyr Val Ala Asp Lys 450 455 460 GAT AGC ATT TCA CAA GAA ATATTT GAA AAT AAA ATT ATT ACA GAT GAG 1440 Asp Ser Ile Ser Gln Glu Ile PheGlu Asn Lys Ile Ile Thr Asp Glu 465 470 475 480 ACT AAT GTA CAA AAT TATTCA GAT AAT TTT TCA TTA GAT GAA TCT ATT 1488 Thr Asn Val Gln Asn Tyr SerAsp Asn Phe Ser Leu Asp Glu Ser Ile 485 490 495 TTA GAT GGG CAA GTT CCTATT AAT CCT GAA ATA GTA GAT CCA CTA TTA 1536 Leu Asp Gly Gln Val Pro IleAsn Pro Glu Ile Val Asp Pro Leu Leu 500 505 510 CCC AAT GTT AAT ATG GAACCT TTA AAT CTT CCA GGT GAA GAA ATA GTA 1584 Pro Asn Val Asn Met Glu ProLeu Asn Leu Pro Gly Glu Glu Ile Val 515 520 525 TTT TAT GAT GAT ATT ACTAAA TAT GTT GAT TAT TTA AAT TCT TAT TAT 1632 Phe Tyr Asp Asp Ile Thr LysTyr Val Asp Tyr Leu Asn Ser Tyr Tyr 530 535 540 TAT TTG GAA TCT CAA AAATTA AGT AAT AAT GTT GAA AAT ATT ACT CTT 1680 Tyr Leu Glu Ser Gln Lys LeuSer Asn Asn Val Glu Asn Ile Thr Leu 545 550 555 560 ACA ACT TCA GTT GAAGAA GCA TTA GGT TAT AGC AAT AAG ATA TAC ACA 1728 Thr Thr Ser Val Glu GluAla Leu Gly Tyr Ser Asn Lys Ile Tyr Thr 565 570 575 TTT TTA CCT AGC TTAGCT GAA AAA GTG AAT AAA GGT GTT CAA GCA GGT 1776 Phe Leu Pro Ser Leu AlaGlu Lys Val Asn Lys Gly Val Gln Ala Gly 580 585 590 TTA TTC TTA AAT TGGGCG AAT GAA GTA GTT GAG GAT TTT ACT ACA AAT 1824 Leu Phe Leu Asn Trp AlaAsn Glu Val Val Glu Asp Phe Thr Thr Asn 595 600 605 ATT ATG AAG AAA GATACA TTG GAT AAA ATA TCA GAT GTA TCA GTA ATA 1872 Ile Met Lys Lys Asp ThrLeu Asp Lys Ile Ser Asp Val Ser Val Ile 610 615 620 ATT CCA TAT ATA GGACCT GCC TTA AAT ATA GGA AAT TCA GCA TTA AGG 1920 Ile Pro Tyr Ile Gly ProAla Leu Asn Ile Gly Asn Ser Ala Leu Arg 625 630 635 640 GGA AAT TTT AAGCAA GCA TTT GCA ACA GCT GGT GTA GCT TTT TTA TTA 1968 Gly Asn Phe Lys GlnAla Phe Ala Thr Ala Gly Val Ala Phe Leu Leu 645 650 655 GAG GGA TTT CCAGAG TTT ACT ATA CCT GCA CTC GGT GTA TTT ACC TTT 2016 Glu Gly Phe Pro GluPhe Thr Ile Pro Ala Leu Gly Val Phe Thr Phe 660 665 670 TAT AGT TCT ATTCAA GAA AGA GAG AAA ATT ATT AAA ACT ATA GAA AAT 2064 Tyr Ser Ser Ile GlnGlu Arg Glu Lys Ile Ile Lys Thr Ile Glu Asn 675 680 685 TGT TTG GAA CAAAGA GTT AAG AGA TGG AAA GAT TCA TAT CAA TGG ATG 2112 Cys Leu Glu Gln ArgVal Lys Arg Trp Lys Asp Ser Tyr Gln Trp Met 690 695 700 GTA TCA AAT TGGTTG TCA AGA ATT ACT ACT CAA TTT AAT CAT ATA AAT 2160 Val Ser Asn Trp LeuSer Arg Ile Thr Thr Gln Phe Asn His Ile Asn 705 710 715 720 TAT CAA ATGTAT GAT TCT TTA AGT TAT CAG GCA GAT GCA ATC AAA GCT 2208 Tyr Gln Met TyrAsp Ser Leu Ser Tyr Gln Ala Asp Ala Ile Lys Ala 725 730 735 AAA ATA GATTTA GAA TAT AAA AAA TAC TCA GGA AGT GAT AAA GAA AAT 2256 Lys Ile Asp LeuGlu Tyr Lys Lys Tyr Ser Gly Ser Asp Lys Glu Asn 740 745 750 ATA AAA AGTCAA GTT GAA AAT TTA AAA AAT AGT TTA GAT GTA AAA ATT 2304 Ile Lys Ser GlnVal Glu Asn Leu Lys Asn Ser Leu Asp Val Lys Ile 755 760 765 TCG GAA GCAATG AAT AAT ATA AAT AAA TTT ATA CGA GAA TGT TCT GTA 2352 Ser Glu Ala MetAsn Asn Ile Asn Lys Phe Ile Arg Glu Cys Ser Val 770 775 780 ACA TAC TTATTT AAA AAT ATG CTC CCT AAA GTA ATT GAC GAA TTA AAT 2400 Thr Tyr Leu PheLys Asn Met Leu Pro Lys Val Ile Asp Glu Leu Asn 785 790 795 800 AAG TTTGAT TTA AGA ACT AAA ACA GAA TTA ATT AAT CTT ATA GAT AGT 2448 Lys Phe AspLeu Arg Thr Lys Thr Glu Leu Ile Asn Leu Ile Asp Ser 805 810 815 CAT AATATT ATT CTA GTT GGT GAA GTA GAT AGA TTA AAA GCA AAA GTA 2496 His Asn IleIle Leu Val Gly Glu Val Asp Arg Leu Lys Ala Lys Val 820 825 830 AAT GAGAGT TTT GAA AAT ACA ATG CCT TTT AAT ATT TTT TCA TAT ACT 2544 Asn Glu SerPhe Glu Asn Thr Met Pro Phe Asn Ile Phe Ser Tyr Thr 835 840 845 AAT AATTCT TTA TTA AAA GAT ATA ATT AAT GAA TAT TTC AAT AGT ATT 2592 Asn Asn SerLeu Leu Lys Asp Ile Ile Asn Glu Tyr Phe Asn Ser Ile 850 855 860 AAT GATTCA AAA ATT TTG AGC TTA CAA AAC AAA AAA AAT GCT TTA GTG 2640 Asn Asp SerLys Ile Leu Ser Leu Gln Asn Lys Lys Asn Ala Leu Val 865 870 875 880 GATACA TCA GGA TAT AAT GCA GAA GTG AGG GTA GGA GAT AAT GTT CAA 2688 Asp ThrSer Gly Tyr Asn Ala Glu Val Arg Val Gly Asp Asn Val Gln 885 890 895 CTTAAT ACG ATA TAT ACA AAT GAC TTT AAA TTA AGT AGT TCA GGA GAT 2736 Leu AsnThr Ile Tyr Thr Asn Asp Phe Lys Leu Ser Ser Ser Gly Asp 900 905 910 AAAATT ATA GTA AAT TTA AAT AAT AAT ATT TTA TAT AGC GCT ATT TAT 2784 Lys IleIle Val Asn Leu Asn Asn Asn Ile Leu Tyr Ser Ala Ile Tyr 915 920 925 GAGAAC TCT AGT GTT AGT TTT TGG ATT AAG ATA TCT AAA GAT TTA ACT 2832 Glu AsnSer Ser Val Ser Phe Trp Ile Lys Ile Ser Lys Asp Leu Thr 930 935 940 AATTCT CAT AAT GAA TAT ACA ATA ATT AAC AGT ATA GAA CAA AAT TCT 2880 Asn SerHis Asn Glu Tyr Thr Ile Ile Asn Ser Ile Glu Gln Asn Ser 945 950 955 960GGG TGG AAA TTA TGT ATT AGG AAT GGC AAT ATA GAA TGG ATT TTA CAA 2928 GlyTrp Lys Leu Cys Ile Arg Asn Gly Asn Ile Glu Trp Ile Leu Gln 965 970 975GAT GTT AAT AGA AAG TAT AAA AGT TTA ATT TTT GAT TAT AGT GAA TCA 2976 AspVal Asn Arg Lys Tyr Lys Ser Leu Ile Phe Asp Tyr Ser Glu Ser 980 985 990TTA AGT CAT ACA GGA TAT ACA AAT AAA TGG TTT TTT GTT ACT ATA ACT 3024 LeuSer His Thr Gly Tyr Thr Asn Lys Trp Phe Phe Val Thr Ile Thr 995 10001005 AAT AAT ATA ATG GGG TAT ATG AAA CTT TAT ATA AAT GGA GAA TTA AAG3072 Asn Asn Ile Met Gly Tyr Met Lys Leu Tyr Ile Asn Gly Glu Leu Lys1010 1015 1020 CAG AGT CAA AAA ATT GAA GAT TTA GAT GAG GTT AAG TTA GATAAA ACC 3120 Gln Ser Gln Lys Ile Glu Asp Leu Asp Glu Val Lys Leu Asp LysThr 1025 1030 1035 1040 ATA GTA TTT GGA ATA GAT GAG AAT ATA GAT GAG AATCAG ATG CTT TGG 3168 Ile Val Phe Gly Ile Asp Glu Asn Ile Asp Glu Asn GlnMet Leu Trp 1045 1050 1055 ATT AGA GAT TTT AAT ATT TTT TCT AAA GAA TTAAGT AAT GAA GAT ATT 3216 Ile Arg Asp Phe Asn Ile Phe Ser Lys Glu Leu SerAsn Glu Asp Ile 1060 1065 1070 AAT ATT GTA TAT GAG GGA CAA ATA TTA AGAAAT GTT ATT AAA GAT TAT 3264 Asn Ile Val Tyr Glu Gly Gln Ile Leu Arg AsnVal Ile Lys Asp Tyr 1075 1080 1085 TGG GGA AAT CCT TTG AAG TTT GAT ACAGAA TAT TAT ATT ATT AAT GAT 3312 Trp Gly Asn Pro Leu Lys Phe Asp Thr GluTyr Tyr Ile Ile Asn Asp 1090 1095 1100 AAT TAT ATA GAT AGG TAT ATT GCACCT GAA AGT AAT GTA CTT GTA CTT 3360 Asn Tyr Ile Asp Arg Tyr Ile Ala ProGlu Ser Asn Val Leu Val Leu 1105 1110 1115 1120 GTT CGG TAT CCA GAT AGATCT AAA TTA TAT ACT GGA AAT CCT ATT ACT 3408 Val Arg Tyr Pro Asp Arg SerLys Leu Tyr Thr Gly Asn Pro Ile Thr 1125 1130 1135 ATT AAA TCA GTA TCTGAT AAG AAT CCT TAT AGT AGA ATT TTA AAT GGA 3456 Ile Lys Ser Val Ser AspLys Asn Pro Tyr Ser Arg Ile Leu Asn Gly 1140 1145 1150 GAT AAT ATA ATTCTT CAT ATG TTA TAT AAT AGT AGG AAA TAT ATG ATA 3504 Asp Asn Ile Ile LeuHis Met Leu Tyr Asn Ser Arg Lys Tyr Met Ile 1155 1160 1165 ATA AGA GATACT GAT ACA ATA TAT GCA ACA CAA GGA GGA GAG TGT TCA 3552 Ile Arg Asp ThrAsp Thr Ile Tyr Ala Thr Gln Gly Gly Glu Cys Ser 1170 1175 1180 CAA AATTGT GTA TAT GCA TTA AAA TTA CAG AGT AAT TTA GGT AAT TAT 3600 Gln Asn CysVal Tyr Ala Leu Lys Leu Gln Ser Asn Leu Gly Asn Tyr 1185 1190 1195 1200GGT ATA GGT ATA TTT AGT ATA AAA AAT ATT GTA TCT AAA AAT AAA TAT 3648 GlyIle Gly Ile Phe Ser Ile Lys Asn Ile Val Ser Lys Asn Lys Tyr 1205 12101215 TGT AGT CAA ATT TTC TCT AGT TTT AGG GAA AAT ACA ATG CTT CTA GCA3696 Cys Ser Gln Ile Phe Ser Ser Phe Arg Glu Asn Thr Met Leu Leu Ala1220 1225 1230 GAT ATA TAT AAA CCT TGG AGA TTT TCT TTT AAA AAT GCA TACACG CCA 3744 Asp Ile Tyr Lys Pro Trp Arg Phe Ser Phe Lys Asn Ala Tyr ThrPro 1235 1240 1245 GTT GCA GTA ACT AAT TAT GAA ACA AAA CTA TTA TCA ACTTCA TCT TTT 3792 Val Ala Val Thr Asn Tyr Glu Thr Lys Leu Leu Ser Thr SerSer Phe 1250 1255 1260 TGG AAA TTT ATT TCT AGG GAT CCA GGA TGG GTA GAGTAA 3831 Trp Lys Phe Ile Ser Arg Asp Pro Gly Trp Val Glu 1265 1270 12751276 amino acids amino acid linear protein 66 Met Thr Trp Pro Val LysAsp Phe Asn Tyr Ser Asp Pro Val Asn Asp 1 5 10 15 Asn Asp Ile Leu TyrLeu Arg Ile Pro Gln Asn Lys Leu Ile Thr Thr 20 25 30 Pro Val Lys Ala PheMet Ile Thr Gln Asn Ile Trp Val Ile Pro Glu 35 40 45 Arg Phe Ser Ser AspThr Asn Pro Ser Leu Ser Lys Pro Pro Arg Pro 50 55 60 Thr Ser Lys Tyr GlnSer Tyr Tyr Asp Pro Ser Tyr Leu Ser Thr Asp 65 70 75 80 Glu Gln Lys AspThr Phe Leu Lys Gly Ile Ile Lys Leu Phe Lys Arg 85 90 95 Ile Asn Glu ArgAsp Ile Gly Lys Lys Leu Ile Asn Tyr Leu Val Val 100 105 110 Gly Ser ProPhe Met Gly Asp Ser Ser Thr Pro Glu Asp Thr Phe Asp 115 120 125 Phe ThrArg His Thr Thr Asn Ile Ala Val Glu Lys Phe Glu Asn Gly 130 135 140 SerTrp Lys Val Thr Asn Ile Ile Thr Pro Ser Val Leu Ile Phe Gly 145 150 155160 Pro Leu Pro Asn Ile Leu Asp Tyr Thr Ala Ser Leu Thr Leu Gln Gly 165170 175 Gln Gln Ser Asn Pro Ser Phe Glu Gly Phe Gly Thr Leu Ser Ile Leu180 185 190 Lys Val Ala Pro Glu Phe Leu Leu Thr Phe Ser Asp Val Thr SerAsn 195 200 205 Gln Ser Ser Ala Val Leu Gly Lys Ser Ile Phe Cys Met AspPro Val 210 215 220 Ile Ala Leu Met His Glu Leu Thr His Ser Leu His GlnLeu Tyr Gly 225 230 235 240 Ile Asn Ile Pro Ser Asp Lys Arg Ile Arg ProGln Val Ser Glu Gly 245 250 255 Phe Phe Ser Gln Asp Gly Pro Asn Val GlnPhe Glu Glu Leu Tyr Thr 260 265 270 Phe Gly Gly Leu Asp Val Glu Ile IlePro Gln Ile Glu Arg Ser Gln 275 280 285 Leu Arg Glu Lys Ala Leu Gly HisTyr Lys Asp Ile Ala Lys Arg Leu 290 295 300 Asn Asn Ile Asn Lys Thr IlePro Ser Ser Trp Ile Ser Asn Ile Asp 305 310 315 320 Lys Tyr Lys Lys IlePhe Ser Glu Lys Tyr Asn Phe Asp Lys Asp Asn 325 330 335 Thr Gly Asn PheVal Val Asn Ile Asp Lys Phe Asn Ser Leu Tyr Ser 340 345 350 Asp Leu ThrAsn Val Met Ser Glu Val Val Tyr Ser Ser Gln Tyr Asn 355 360 365 Val LysAsn Arg Thr His Tyr Phe Ser Arg His Tyr Leu Pro Val Phe 370 375 380 AlaAsn Ile Leu Asp Asp Asn Ile Tyr Thr Ile Arg Asp Gly Phe Asn 385 390 395400 Leu Thr Asn Lys Gly Phe Asn Ile Glu Asn Ser Gly Gln Asn Ile Glu 405410 415 Arg Asn Pro Ala Leu Gln Lys Leu Ser Ser Glu Ser Val Val Asp Leu420 425 430 Phe Thr Lys Val Cys Leu Arg Leu Thr Lys Asn Ser Arg Asp AspSer 435 440 445 Thr Cys Ile Lys Val Lys Asn Asn Arg Leu Pro Tyr Val AlaAsp Lys 450 455 460 Asp Ser Ile Ser Gln Glu Ile Phe Glu Asn Lys Ile IleThr Asp Glu 465 470 475 480 Thr Asn Val Gln Asn Tyr Ser Asp Asn Phe SerLeu Asp Glu Ser Ile 485 490 495 Leu Asp Gly Gln Val Pro Ile Asn Pro GluIle Val Asp Pro Leu Leu 500 505 510 Pro Asn Val Asn Met Glu Pro Leu AsnLeu Pro Gly Glu Glu Ile Val 515 520 525 Phe Tyr Asp Asp Ile Thr Lys TyrVal Asp Tyr Leu Asn Ser Tyr Tyr 530 535 540 Tyr Leu Glu Ser Gln Lys LeuSer Asn Asn Val Glu Asn Ile Thr Leu 545 550 555 560 Thr Thr Ser Val GluGlu Ala Leu Gly Tyr Ser Asn Lys Ile Tyr Thr 565 570 575 Phe Leu Pro SerLeu Ala Glu Lys Val Asn Lys Gly Val Gln Ala Gly 580 585 590 Leu Phe LeuAsn Trp Ala Asn Glu Val Val Glu Asp Phe Thr Thr Asn 595 600 605 Ile MetLys Lys Asp Thr Leu Asp Lys Ile Ser Asp Val Ser Val Ile 610 615 620 IlePro Tyr Ile Gly Pro Ala Leu Asn Ile Gly Asn Ser Ala Leu Arg 625 630 635640 Gly Asn Phe Lys Gln Ala Phe Ala Thr Ala Gly Val Ala Phe Leu Leu 645650 655 Glu Gly Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly Val Phe Thr Phe660 665 670 Tyr Ser Ser Ile Gln Glu Arg Glu Lys Ile Ile Lys Thr Ile GluAsn 675 680 685 Cys Leu Glu Gln Arg Val Lys Arg Trp Lys Asp Ser Tyr GlnTrp Met 690 695 700 Val Ser Asn Trp Leu Ser Arg Ile Thr Thr Gln Phe AsnHis Ile Asn 705 710 715 720 Tyr Gln Met Tyr Asp Ser Leu Ser Tyr Gln AlaAsp Ala Ile Lys Ala 725 730 735 Lys Ile Asp Leu Glu Tyr Lys Lys Tyr SerGly Ser Asp Lys Glu Asn 740 745 750 Ile Lys Ser Gln Val Glu Asn Leu LysAsn Ser Leu Asp Val Lys Ile 755 760 765 Ser Glu Ala Met Asn Asn Ile AsnLys Phe Ile Arg Glu Cys Ser Val 770 775 780 Thr Tyr Leu Phe Lys Asn MetLeu Pro Lys Val Ile Asp Glu Leu Asn 785 790 795 800 Lys Phe Asp Leu ArgThr Lys Thr Glu Leu Ile Asn Leu Ile Asp Ser 805 810 815 His Asn Ile IleLeu Val Gly Glu Val Asp Arg Leu Lys Ala Lys Val 820 825 830 Asn Glu SerPhe Glu Asn Thr Met Pro Phe Asn Ile Phe Ser Tyr Thr 835 840 845 Asn AsnSer Leu Leu Lys Asp Ile Ile Asn Glu Tyr Phe Asn Ser Ile 850 855 860 AsnAsp Ser Lys Ile Leu Ser Leu Gln Asn Lys Lys Asn Ala Leu Val 865 870 875880 Asp Thr Ser Gly Tyr Asn Ala Glu Val Arg Val Gly Asp Asn Val Gln 885890 895 Leu Asn Thr Ile Tyr Thr Asn Asp Phe Lys Leu Ser Ser Ser Gly Asp900 905 910 Lys Ile Ile Val Asn Leu Asn Asn Asn Ile Leu Tyr Ser Ala IleTyr 915 920 925 Glu Asn Ser Ser Val Ser Phe Trp Ile Lys Ile Ser Lys AspLeu Thr 930 935 940 Asn Ser His Asn Glu Tyr Thr Ile Ile Asn Ser Ile GluGln Asn Ser 945 950 955 960 Gly Trp Lys Leu Cys Ile Arg Asn Gly Asn IleGlu Trp Ile Leu Gln 965 970 975 Asp Val Asn Arg Lys Tyr Lys Ser Leu IlePhe Asp Tyr Ser Glu Ser 980 985 990 Leu Ser His Thr Gly Tyr Thr Asn LysTrp Phe Phe Val Thr Ile Thr 995 1000 1005 Asn Asn Ile Met Gly Tyr MetLys Leu Tyr Ile Asn Gly Glu Leu Lys 1010 1015 1020 Gln Ser Gln Lys IleGlu Asp Leu Asp Glu Val Lys Leu Asp Lys Thr 1025 1030 1035 1040 Ile ValPhe Gly Ile Asp Glu Asn Ile Asp Glu Asn Gln Met Leu Trp 1045 1050 1055Ile Arg Asp Phe Asn Ile Phe Ser Lys Glu Leu Ser Asn Glu Asp Ile 10601065 1070 Asn Ile Val Tyr Glu Gly Gln Ile Leu Arg Asn Val Ile Lys AspTyr 1075 1080 1085 Trp Gly Asn Pro Leu Lys Phe Asp Thr Glu Tyr Tyr IleIle Asn Asp 1090 1095 1100 Asn Tyr Ile Asp Arg Tyr Ile Ala Pro Glu SerAsn Val Leu Val Leu 1105 1110 1115 1120 Val Arg Tyr Pro Asp Arg Ser LysLeu Tyr Thr Gly Asn Pro Ile Thr 1125 1130 1135 Ile Lys Ser Val Ser AspLys Asn Pro Tyr Ser Arg Ile Leu Asn Gly 1140 1145 1150 Asp Asn Ile IleLeu His Met Leu Tyr Asn Ser Arg Lys Tyr Met Ile 1155 1160 1165 Ile ArgAsp Thr Asp Thr Ile Tyr Ala Thr Gln Gly Gly Glu Cys Ser 1170 1175 1180Gln Asn Cys Val Tyr Ala Leu Lys Leu Gln Ser Asn Leu Gly Asn Tyr 11851190 1195 1200 Gly Ile Gly Ile Phe Ser Ile Lys Asn Ile Val Ser Lys AsnLys Tyr 1205 1210 1215 Cys Ser Gln Ile Phe Ser Ser Phe Arg Glu Asn ThrMet Leu Leu Ala 1220 1225 1230 Asp Ile Tyr Lys Pro Trp Arg Phe Ser PheLys Asn Ala Tyr Thr Pro 1235 1240 1245 Val Ala Val Thr Asn Tyr Glu ThrLys Leu Leu Ser Thr Ser Ser Phe 1250 1255 1260 Trp Lys Phe Ile Ser ArgAsp Pro Gly Trp Val Glu 1265 1270 1275 1469 base pairs nucleic aciddouble linear DNA (genomic) CDS 108..1460 67 AGATCTCGAT CCCGCGAAATTAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60 TTCCCCTCTA GAAATAATTTTGTTTAACTT TAAGAAGGAG ATATACC ATG GGC CAT 116 Met Gly His 1 CAT CAT CATCAT CAT CAT CAT CAT CAC AGC AGC GGC CAT ATC GAA GGT 164 His His His HisHis His His His His Ser Ser Gly His Ile Glu Gly 5 10 15 CGT CAT ATG GCTAGC ATG GCT TTA TTA AAA GAT ATA ATT AAT GAA TAT 212 Arg His Met Ala SerMet Ala Leu Leu Lys Asp Ile Ile Asn Glu Tyr 20 25 30 35 TTC AAT AGT ATTAAT GAT TCA AAA ATT TTG AGC TTA CAA AAC AAA AAA 260 Phe Asn Ser Ile AsnAsp Ser Lys Ile Leu Ser Leu Gln Asn Lys Lys 40 45 50 AAT GCT TTA GTG GATACA TCA GGA TAT AAT GCA GAA GTG AGG GTA GGA 308 Asn Ala Leu Val Asp ThrSer Gly Tyr Asn Ala Glu Val Arg Val Gly 55 60 65 GAT AAT GTT CAA CTT AATACG ATA TAT ACA AAT GAC TTT AAA TTA AGT 356 Asp Asn Val Gln Leu Asn ThrIle Tyr Thr Asn Asp Phe Lys Leu Ser 70 75 80 AGT TCA GGA GAT AAA ATT ATAGTA AAT TTA AAT AAT AAT ATT TTA TAT 404 Ser Ser Gly Asp Lys Ile Ile ValAsn Leu Asn Asn Asn Ile Leu Tyr 85 90 95 AGC GCT ATT TAT GAG AAC TCT AGTGTT AGT TTT TGG ATT AAG ATA TCT 452 Ser Ala Ile Tyr Glu Asn Ser Ser ValSer Phe Trp Ile Lys Ile Ser 100 105 110 115 AAA GAT TTA ACT AAT TCT CATAAT GAA TAT ACA ATA ATT AAC AGT ATA 500 Lys Asp Leu Thr Asn Ser His AsnGlu Tyr Thr Ile Ile Asn Ser Ile 120 125 130 GAA CAA AAT TCT GGG TGG AAATTA TGT ATT AGG AAT GGC AAT ATA GAA 548 Glu Gln Asn Ser Gly Trp Lys LeuCys Ile Arg Asn Gly Asn Ile Glu 135 140 145 TGG ATT TTA CAA GAT GTT AATAGA AAG TAT AAA AGT TTA ATT TTT GAT 596 Trp Ile Leu Gln Asp Val Asn ArgLys Tyr Lys Ser Leu Ile Phe Asp 150 155 160 TAT AGT GAA TCA TTA AGT CATACA GGA TAT ACA AAT AAA TGG TTT TTT 644 Tyr Ser Glu Ser Leu Ser His ThrGly Tyr Thr Asn Lys Trp Phe Phe 165 170 175 GTT ACT ATA ACT AAT AAT ATAATG GGG TAT ATG AAA CTT TAT ATA AAT 692 Val Thr Ile Thr Asn Asn Ile MetGly Tyr Met Lys Leu Tyr Ile Asn 180 185 190 195 GGA GAA TTA AAG CAG AGTCAA AAA ATT GAA GAT TTA GAT GAG GTT AAG 740 Gly Glu Leu Lys Gln Ser GlnLys Ile Glu Asp Leu Asp Glu Val Lys 200 205 210 TTA GAT AAA ACC ATA GTATTT GGA ATA GAT GAG AAT ATA GAT GAG AAT 788 Leu Asp Lys Thr Ile Val PheGly Ile Asp Glu Asn Ile Asp Glu Asn 215 220 225 CAG ATG CTT TGG ATT AGAGAT TTT AAT ATT TTT TCT AAA GAA TTA AGT 836 Gln Met Leu Trp Ile Arg AspPhe Asn Ile Phe Ser Lys Glu Leu Ser 230 235 240 AAT GAA GAT ATT AAT ATTGTA TAT GAG GGA CAA ATA TTA AGA AAT GTT 884 Asn Glu Asp Ile Asn Ile ValTyr Glu Gly Gln Ile Leu Arg Asn Val 245 250 255 ATT AAA GAT TAT TGG GGAAAT CCT TTG AAG TTT GAT ACA GAA TAT TAT 932 Ile Lys Asp Tyr Trp Gly AsnPro Leu Lys Phe Asp Thr Glu Tyr Tyr 260 265 270 275 ATT ATT AAT GAT AATTAT ATA GAT AGG TAT ATT GCA CCT GAA AGT AAT 980 Ile Ile Asn Asp Asn TyrIle Asp Arg Tyr Ile Ala Pro Glu Ser Asn 280 285 290 GTA CTT GTA CTT GTTCGG TAT CCA GAT AGA TCT AAA TTA TAT ACT GGA 1028 Val Leu Val Leu Val ArgTyr Pro Asp Arg Ser Lys Leu Tyr Thr Gly 295 300 305 AAT CCT ATT ACT ATTAAA TCA GTA TCT GAT AAG AAT CCT TAT AGT AGA 1076 Asn Pro Ile Thr Ile LysSer Val Ser Asp Lys Asn Pro Tyr Ser Arg 310 315 320 ATT TTA AAT GGA GATAAT ATA ATT CTT CAT ATG TTA TAT AAT AGT AGG 1124 Ile Leu Asn Gly Asp AsnIle Ile Leu His Met Leu Tyr Asn Ser Arg 325 330 335 AAA TAT ATG ATA ATAAGA GAT ACT GAT ACA ATA TAT GCA ACA CAA GGA 1172 Lys Tyr Met Ile Ile ArgAsp Thr Asp Thr Ile Tyr Ala Thr Gln Gly 340 345 350 355 GGA GAG TGT TCACAA AAT TGT GTA TAT GCA TTA AAA TTA CAG AGT AAT 1220 Gly Glu Cys Ser GlnAsn Cys Val Tyr Ala Leu Lys Leu Gln Ser Asn 360 365 370 TTA GGT AAT TATGGT ATA GGT ATA TTT AGT ATA AAA AAT ATT GTA TCT 1268 Leu Gly Asn Tyr GlyIle Gly Ile Phe Ser Ile Lys Asn Ile Val Ser 375 380 385 AAA AAT AAA TATTGT AGT CAA ATT TTC TCT AGT TTT AGG GAA AAT ACA 1316 Lys Asn Lys Tyr CysSer Gln Ile Phe Ser Ser Phe Arg Glu Asn Thr 390 395 400 ATG CTT CTA GCAGAT ATA TAT AAA CCT TGG AGA TTT TCT TTT AAA AAT 1364 Met Leu Leu Ala AspIle Tyr Lys Pro Trp Arg Phe Ser Phe Lys Asn 405 410 415 GCA TAC ACG CCAGTT GCA GTA ACT AAT TAT GAA ACA AAA CTA TTA TCA 1412 Ala Tyr Thr Pro ValAla Val Thr Asn Tyr Glu Thr Lys Leu Leu Ser 420 425 430 435 ACT TCA TCTTTT TGG AAA TTT ATT TCT AGG GAT CCA GGA TGG GTA GAG 1460 Thr Ser Ser PheTrp Lys Phe Ile Ser Arg Asp Pro Gly Trp Val Glu 440 445 450 TAAAAGCTT1469 451 amino acids amino acid linear protein 68 Met Gly His His HisHis His His His His His His Ser Ser Gly His 1 5 10 15 Ile Glu Gly ArgHis Met Ala Ser Met Ala Leu Leu Lys Asp Ile Ile 20 25 30 Asn Glu Tyr PheAsn Ser Ile Asn Asp Ser Lys Ile Leu Ser Leu Gln 35 40 45 Asn Lys Lys AsnAla Leu Val Asp Thr Ser Gly Tyr Asn Ala Glu Val 50 55 60 Arg Val Gly AspAsn Val Gln Leu Asn Thr Ile Tyr Thr Asn Asp Phe 65 70 75 80 Lys Leu SerSer Ser Gly Asp Lys Ile Ile Val Asn Leu Asn Asn Asn 85 90 95 Ile Leu TyrSer Ala Ile Tyr Glu Asn Ser Ser Val Ser Phe Trp Ile 100 105 110 Lys IleSer Lys Asp Leu Thr Asn Ser His Asn Glu Tyr Thr Ile Ile 115 120 125 AsnSer Ile Glu Gln Asn Ser Gly Trp Lys Leu Cys Ile Arg Asn Gly 130 135 140Asn Ile Glu Trp Ile Leu Gln Asp Val Asn Arg Lys Tyr Lys Ser Leu 145 150155 160 Ile Phe Asp Tyr Ser Glu Ser Leu Ser His Thr Gly Tyr Thr Asn Lys165 170 175 Trp Phe Phe Val Thr Ile Thr Asn Asn Ile Met Gly Tyr Met LysLeu 180 185 190 Tyr Ile Asn Gly Glu Leu Lys Gln Ser Gln Lys Ile Glu AspLeu Asp 195 200 205 Glu Val Lys Leu Asp Lys Thr Ile Val Phe Gly Ile AspGlu Asn Ile 210 215 220 Asp Glu Asn Gln Met Leu Trp Ile Arg Asp Phe AsnIle Phe Ser Lys 225 230 235 240 Glu Leu Ser Asn Glu Asp Ile Asn Ile ValTyr Glu Gly Gln Ile Leu 245 250 255 Arg Asn Val Ile Lys Asp Tyr Trp GlyAsn Pro Leu Lys Phe Asp Thr 260 265 270 Glu Tyr Tyr Ile Ile Asn Asp AsnTyr Ile Asp Arg Tyr Ile Ala Pro 275 280 285 Glu Ser Asn Val Leu Val LeuVal Arg Tyr Pro Asp Arg Ser Lys Leu 290 295 300 Tyr Thr Gly Asn Pro IleThr Ile Lys Ser Val Ser Asp Lys Asn Pro 305 310 315 320 Tyr Ser Arg IleLeu Asn Gly Asp Asn Ile Ile Leu His Met Leu Tyr 325 330 335 Asn Ser ArgLys Tyr Met Ile Ile Arg Asp Thr Asp Thr Ile Tyr Ala 340 345 350 Thr GlnGly Gly Glu Cys Ser Gln Asn Cys Val Tyr Ala Leu Lys Leu 355 360 365 GlnSer Asn Leu Gly Asn Tyr Gly Ile Gly Ile Phe Ser Ile Lys Asn 370 375 380Ile Val Ser Lys Asn Lys Tyr Cys Ser Gln Ile Phe Ser Ser Phe Arg 385 390395 400 Glu Asn Thr Met Leu Leu Ala Asp Ile Tyr Lys Pro Trp Arg Phe Ser405 410 415 Phe Lys Asn Ala Tyr Thr Pro Val Ala Val Thr Asn Tyr Glu ThrLys 420 425 430 Leu Leu Ser Thr Ser Ser Phe Trp Lys Phe Ile Ser Arg AspPro Gly 435 440 445 Trp Val Glu 450 32 base pairs nucleic acid singlelinear other nucleic acid /desc = “DNA” 69 GCAAGCTTTT ACTCTACCCATCCTGGATCC CT 32 3825 base pairs nucleic acid double linear DNA(genomic) CDS 1..3822 70 ATG CCA GTT GCA ATA AAT AGT TTT AAT TAT AAT GACCCT GTT AAT GAT 48 Met Pro Val Ala Ile Asn Ser Phe Asn Tyr Asn Asp ProVal Asn Asp 1 5 10 15 GAT ACA ATT TTA TAC ATG CAG ATA CCA TAT GAA GAAAAA AGT AAA AAA 96 Asp Thr Ile Leu Tyr Met Gln Ile Pro Tyr Glu Glu LysSer Lys Lys 20 25 30 TAT TAT AAA GCT TTT GAG ATT ATG CGT AAT GTT TGG ATAATT CCT GAG 144 Tyr Tyr Lys Ala Phe Glu Ile Met Arg Asn Val Trp Ile IlePro Glu 35 40 45 AGA AAT ACA ATA GGA ACG AAT CCT AGT GAT TTT GAT CCA CCGGCT TCA 192 Arg Asn Thr Ile Gly Thr Asn Pro Ser Asp Phe Asp Pro Pro AlaSer 50 55 60 TTA AAG AAC GGA AGC AGT GCT TAT TAT GAT CCT AAT TAT TTA ACCACT 240 Leu Lys Asn Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Leu Thr Thr65 70 75 80 GAT GCT GAA AAA GAT AGA TAT TTA AAA ACA ACG ATA AAA TTA TTTAAG 288 Asp Ala Glu Lys Asp Arg Tyr Leu Lys Thr Thr Ile Lys Leu Phe Lys85 90 95 AGA ATT AAT AGT AAT CCT GCA GGG AAA GTT TTG TTA CAA GAA ATA TCA336 Arg Ile Asn Ser Asn Pro Ala Gly Lys Val Leu Leu Gln Glu Ile Ser 100105 110 TAT GCT AAA CCA TAT TTA GGA AAT GAC CAC ACG CCA ATT GAT GAA TTC384 Tyr Ala Lys Pro Tyr Leu Gly Asn Asp His Thr Pro Ile Asp Glu Phe 115120 125 TCT CCA GTT ACT AGA ACT ACA AGT GTT AAT ATA AAA TTA TCA ACT AAT432 Ser Pro Val Thr Arg Thr Thr Ser Val Asn Ile Lys Leu Ser Thr Asn 130135 140 GTT GAA AGT TCA ATG TTA TTG AAT CTT CTT GTA TTG GGA GCA GGA CCT480 Val Glu Ser Ser Met Leu Leu Asn Leu Leu Val Leu Gly Ala Gly Pro 145150 155 160 GAT ATA TTT GAA AGT TGT TGT TAC CCC GTT AGA AAA CTA ATA GATCCA 528 Asp Ile Phe Glu Ser Cys Cys Tyr Pro Val Arg Lys Leu Ile Asp Pro165 170 175 GAT GTA GTT TAT GAT CCA AGT AAT TAT GGT TTT GGA TCA ATT AATATC 576 Asp Val Val Tyr Asp Pro Ser Asn Tyr Gly Phe Gly Ser Ile Asn Ile180 185 190 GTG ACA TTT TCA CCT GAG TAT GAA TAT ACT TTT AAT GAT ATT AGTGGA 624 Val Thr Phe Ser Pro Glu Tyr Glu Tyr Thr Phe Asn Asp Ile Ser Gly195 200 205 GGG CAT AAT AGT AGT ACA GAA TCA TTT ATT GCA GAT CCT GCA ATTTCA 672 Gly His Asn Ser Ser Thr Glu Ser Phe Ile Ala Asp Pro Ala Ile Ser210 215 220 CTA GCT CAT GAA TTG ATA CAT GCA CTG CAT GGA TTA TAC GGG GCTAGG 720 Leu Ala His Glu Leu Ile His Ala Leu His Gly Leu Tyr Gly Ala Arg225 230 235 240 GGA GTT ACT TAT GAA GAG ACT ATA GAA GTA AAG CAA GCA CCTCTT ATG 768 Gly Val Thr Tyr Glu Glu Thr Ile Glu Val Lys Gln Ala Pro LeuMet 245 250 255 ATA GCC GAA AAA CCC ATA AGG CTA GAA GAA TTT TTA ACC TTTGGA GGT 816 Ile Ala Glu Lys Pro Ile Arg Leu Glu Glu Phe Leu Thr Phe GlyGly 260 265 270 CAG GAT TTA AAT ATT ATT ACT AGT GCT ATG AAG GAA AAA ATATAT AAC 864 Gln Asp Leu Asn Ile Ile Thr Ser Ala Met Lys Glu Lys Ile TyrAsn 275 280 285 AAT CTT TTA GCT AAC TAT GAA AAA ATA GCT ACT AGA CTT AGTGAA GTT 912 Asn Leu Leu Ala Asn Tyr Glu Lys Ile Ala Thr Arg Leu Ser GluVal 290 295 300 AAT AGT GCT CCT CCT GAA TAT GAT ATT AAT GAA TAT AAA GATTAT TTT 960 Asn Ser Ala Pro Pro Glu Tyr Asp Ile Asn Glu Tyr Lys Asp TyrPhe 305 310 315 320 CAA TGG AAG TAT GGG CTA GAT AAA AAT GCT GAT GGA AGTTAT ACT GTA 1008 Gln Trp Lys Tyr Gly Leu Asp Lys Asn Ala Asp Gly Ser TyrThr Val 325 330 335 AAT GAA AAT AAA TTT AAT GAA ATT TAT AAA AAA TTA TATAGT TTT ACA 1056 Asn Glu Asn Lys Phe Asn Glu Ile Tyr Lys Lys Leu Tyr SerPhe Thr 340 345 350 GAG AGT GAC TTA GCA AAT AAA TTT AAA GTA AAA TGT AGAAAT ACT TAT 1104 Glu Ser Asp Leu Ala Asn Lys Phe Lys Val Lys Cys Arg AsnThr Tyr 355 360 365 TTT ATT AAA TAT GAA TTT TTA AAA GTT CCA AAT TTG TTAGAT GAT GAT 1152 Phe Ile Lys Tyr Glu Phe Leu Lys Val Pro Asn Leu Leu AspAsp Asp 370 375 380 ATT TAT ACT GTA TCA GAG GGG TTT AAT ATA GGT AAT TTAGCA GTA AAC 1200 Ile Tyr Thr Val Ser Glu Gly Phe Asn Ile Gly Asn Leu AlaVal Asn 385 390 395 400 AAT CGC GGA CAA AGT ATA AAG TTA AAT CCT AAA ATTATT GAT TCC ATT 1248 Asn Arg Gly Gln Ser Ile Lys Leu Asn Pro Lys Ile IleAsp Ser Ile 405 410 415 CCA GAT AAA GGT CTA GTA GAA AAG ATC GTT AAA TTTTGT AAG AGC GTT 1296 Pro Asp Lys Gly Leu Val Glu Lys Ile Val Lys Phe CysLys Ser Val 420 425 430 ATT CCT AGA AAA GGT ACA AAG GCG CCA CCG CGA CTATGC ATT AGA GTA 1344 Ile Pro Arg Lys Gly Thr Lys Ala Pro Pro Arg Leu CysIle Arg Val 435 440 445 AAT AAT AGT GAG TTA TTT TTT GTA GCT TCA GAA AGTAGC TAT AAT GAA 1392 Asn Asn Ser Glu Leu Phe Phe Val Ala Ser Glu Ser SerTyr Asn Glu 450 455 460 AAT GAT ATT AAT ACA CCT AAA GAA ATT GAC GAT ACAACA AAT CTA AAT 1440 Asn Asp Ile Asn Thr Pro Lys Glu Ile Asp Asp Thr ThrAsn Leu Asn 465 470 475 480 AAT AAT TAT AGA AAT AAT TTA GAT GAA GTT ATTTTA GAT TAT AAT AGT 1488 Asn Asn Tyr Arg Asn Asn Leu Asp Glu Val Ile LeuAsp Tyr Asn Ser 485 490 495 CAG ACA ATA CCT CAA ATA TCA AAT CGA ACA TTAAAT ACA CTT GTA CAA 1536 Gln Thr Ile Pro Gln Ile Ser Asn Arg Thr Leu AsnThr Leu Val Gln 500 505 510 GAC AAT AGT TAT GTG CCA AGA TAT GAT TCT AATGGA ACA AGT GAA ATA 1584 Asp Asn Ser Tyr Val Pro Arg Tyr Asp Ser Asn GlyThr Ser Glu Ile 515 520 525 GAG GAA TAT GAT GTT GTT GAC TTT AAT GTA TTTTTC TAT TTA CAT GCA 1632 Glu Glu Tyr Asp Val Val Asp Phe Asn Val Phe PheTyr Leu His Ala 530 535 540 CAA AAA GTG CCA GAA GGT GAA ACC AAT ATA AGTTTA ACT TCT TCA ATT 1680 Gln Lys Val Pro Glu Gly Glu Thr Asn Ile Ser LeuThr Ser Ser Ile 545 550 555 560 GAT ACA GCA TTA TTA GAA GAA TCC AAA GATATA TTT TTT TCT TCA GAG 1728 Asp Thr Ala Leu Leu Glu Glu Ser Lys Asp IlePhe Phe Ser Ser Glu 565 570 575 TTT ATC GAT ACT ATC AAT AAA CCT GTA AATGCA GCA CTA TTT ATA GAT 1776 Phe Ile Asp Thr Ile Asn Lys Pro Val Asn AlaAla Leu Phe Ile Asp 580 585 590 TGG ATA AGC AAA GTA ATA AGA GAT TTT ACCACT GAA GCT ACA CAA AAA 1824 Trp Ile Ser Lys Val Ile Arg Asp Phe Thr ThrGlu Ala Thr Gln Lys 595 600 605 AGT ACT GTT GAT AAG ATT GCA GAC ATA TCTTTA ATT GTA CCC TAT GTA 1872 Ser Thr Val Asp Lys Ile Ala Asp Ile Ser LeuIle Val Pro Tyr Val 610 615 620 GGT CTT GCT TTG AAT ATA ATT ATT GAG GCAGAA AAA GGA AAT TTT GAG 1920 Gly Leu Ala Leu Asn Ile Ile Ile Glu Ala GluLys Gly Asn Phe Glu 625 630 635 640 GAG GCA TTT GAA TTA TTA GGA GTG GGTATT TTA TTA GAA TTT GTG CCA 1968 Glu Ala Phe Glu Leu Leu Gly Val Gly IleLeu Leu Glu Phe Val Pro 645 650 655 GAA CTT ACA ATT CCT GTA ATT TTA GTGTTT ACG ATA AAA TCC TAT ATA 2016 Glu Leu Thr Ile Pro Val Ile Leu Val PheThr Ile Lys Ser Tyr Ile 660 665 670 GAT TCA TAT GAG AAT AAA AAT AAA GCAATT AAA GCA ATA AAT AAT TCA 2064 Asp Ser Tyr Glu Asn Lys Asn Lys Ala IleLys Ala Ile Asn Asn Ser 675 680 685 TTA ATC GAA AGA GAA GCA AAG TGG AAAGAA ATA TAT AGT TGG ATA GTA 2112 Leu Ile Glu Arg Glu Ala Lys Trp Lys GluIle Tyr Ser Trp Ile Val 690 695 700 TCA AAT TGG CTT ACT AGA ATT AAT ACTCAA TTT AAT AAA AGA AAA GAG 2160 Ser Asn Trp Leu Thr Arg Ile Asn Thr GlnPhe Asn Lys Arg Lys Glu 705 710 715 720 CAA ATG TAT CAG GCT TTA CAA AATCAA GTA GAT GCA ATA AAA ACA GCA 2208 Gln Met Tyr Gln Ala Leu Gln Asn GlnVal Asp Ala Ile Lys Thr Ala 725 730 735 ATA GAA TAT AAA TAT AAT AAT TATACT TCA GAT GAG AAA AAT AGA CTT 2256 Ile Glu Tyr Lys Tyr Asn Asn Tyr ThrSer Asp Glu Lys Asn Arg Leu 740 745 750 GAA TCT GAA TAT AAT ATC AAT AATATA GAA GAA GAA TTG AAT AAA AAA 2304 Glu Ser Glu Tyr Asn Ile Asn Asn IleGlu Glu Glu Leu Asn Lys Lys 755 760 765 GTT TCT TTA GCA ATG AAA AAT ATAGAA AGA TTT ATG ACA GAA AGT TCT 2352 Val Ser Leu Ala Met Lys Asn Ile GluArg Phe Met Thr Glu Ser Ser 770 775 780 ATA TCT TAT TTA ATG AAA TTA ATAAAT GAA GCC AAA GTT GGT AAA TTA 2400 Ile Ser Tyr Leu Met Lys Leu Ile AsnGlu Ala Lys Val Gly Lys Leu 785 790 795 800 AAA AAA TAT GAT AAC CAT GTTAAG AGC GAT TTA TTA AAC TAT ATT CTC 2448 Lys Lys Tyr Asp Asn His Val LysSer Asp Leu Leu Asn Tyr Ile Leu 805 810 815 GAC CAT AGA TCA ATC TTA GGAGAG CAG ACA AAT GAA TTA AGT GAT TTG 2496 Asp His Arg Ser Ile Leu Gly GluGln Thr Asn Glu Leu Ser Asp Leu 820 825 830 GTG ACT AGT ACT TTG AAT AGTAGT ATT CCA TTT GAA CTT TCT TCA TAT 2544 Val Thr Ser Thr Leu Asn Ser SerIle Pro Phe Glu Leu Ser Ser Tyr 835 840 845 ACT AAT GAT AAA ATT CTA ATTATA TAT TTT AAT AGA TTA TAT AAA AAA 2592 Thr Asn Asp Lys Ile Leu Ile IleTyr Phe Asn Arg Leu Tyr Lys Lys 850 855 860 ATT AAA GAT AGT TCT ATT TTAGAT ATG CGA TAT GAA AAT AAT AAA TTT 2640 Ile Lys Asp Ser Ser Ile Leu AspMet Arg Tyr Glu Asn Asn Lys Phe 865 870 875 880 ATA GAT ATC TCT GGA TATGGT TCA AAT ATA AGC ATT AAT GGA AAC GTA 2688 Ile Asp Ile Ser Gly Tyr GlySer Asn Ile Ser Ile Asn Gly Asn Val 885 890 895 TAT ATT TAT TCA ACA AATAGA AAT CAA TTT GGA ATA TAT AAT AGT AGG 2736 Tyr Ile Tyr Ser Thr Asn ArgAsn Gln Phe Gly Ile Tyr Asn Ser Arg 900 905 910 CTT AGT GAA GTT AAT ATAGCT CAA AAT AAT GAT ATT ATA TAC AAT AGT 2784 Leu Ser Glu Val Asn Ile AlaGln Asn Asn Asp Ile Ile Tyr Asn Ser 915 920 925 AGA TAT CAA AAT TTT AGTATT AGT TTC TGG GTA AGG ATT CCT AAA CAC 2832 Arg Tyr Gln Asn Phe Ser IleSer Phe Trp Val Arg Ile Pro Lys His 930 935 940 TAC AAA CCT ATG AAT CATAAT CGG GAA TAC ACT ATA ATA AAT TGT ATG 2880 Tyr Lys Pro Met Asn His AsnArg Glu Tyr Thr Ile Ile Asn Cys Met 945 950 955 960 GGG AAT AAT AAT TCGGGA TGG AAA ATA TCA CTT AGA ACT GTT AGA GAT 2928 Gly Asn Asn Asn Ser GlyTrp Lys Ile Ser Leu Arg Thr Val Arg Asp 965 970 975 TGT GAA ATA ATT TGGACT TTA CAA GAT ACT TCT GGA AAT AAG GAA AAT 2976 Cys Glu Ile Ile Trp ThrLeu Gln Asp Thr Ser Gly Asn Lys Glu Asn 980 985 990 TTA ATT TTT AGG TATGAA GAA CTT AAT AGG ATA TCT AAT TAT ATA AAT 3024 Leu Ile Phe Arg Tyr GluGlu Leu Asn Arg Ile Ser Asn Tyr Ile Asn 995 1000 1005 AAA TGG ATT TTTGTA ACT ATT ACT AAT AAT AGA TTA GGC AAT TCT AGA 3072 Lys Trp Ile Phe ValThr Ile Thr Asn Asn Arg Leu Gly Asn Ser Arg 1010 1015 1020 ATT TAC ATCAAT GGA AAT TTA ATA GTT GAA AAA TCA ATT TCG AAT TTA 3120 Ile Tyr Ile AsnGly Asn Leu Ile Val Glu Lys Ser Ile Ser Asn Leu 1025 1030 1035 1040 GGTGAT ATT CAT GTT AGT GAT AAT ATA TTA TTT AAA ATT GTT GGT TGT 3168 Gly AspIle His Val Ser Asp Asn Ile Leu Phe Lys Ile Val Gly Cys 1045 1050 1055GAT GAT GAA ACG TAT GTT GGT ATA AGA TAT TTT AAA GTT TTT AAT ACG 3216 AspAsp Glu Thr Tyr Val Gly Ile Arg Tyr Phe Lys Val Phe Asn Thr 1060 10651070 GAA TTA GAT AAA ACA GAA ATT GAG ACT TTA TAT AGT AAT GAG CCA GAT3264 Glu Leu Asp Lys Thr Glu Ile Glu Thr Leu Tyr Ser Asn Glu Pro Asp1075 1080 1085 CCA AGT ATC TTA AAA AAC TAT TGG GGA AAT TAT TTG CTA TATAAT AAA 3312 Pro Ser Ile Leu Lys Asn Tyr Trp Gly Asn Tyr Leu Leu Tyr AsnLys 1090 1095 1100 AAA TAT TAT TTA TTC AAT TTA CTA AGA AAA GAT AAG TATATT ACT CTG 3360 Lys Tyr Tyr Leu Phe Asn Leu Leu Arg Lys Asp Lys Tyr IleThr Leu 1105 1110 1115 1120 AAT TCA GGC ATT TTA AAT ATT AAT CAA CAA AGAGGT GTT ACT GAA GGC 3408 Asn Ser Gly Ile Leu Asn Ile Asn Gln Gln Arg GlyVal Thr Glu Gly 1125 1130 1135 TCT GTT TTT TTG AAC TAT AAA TTA TAT GAAGGA GTA GAA GTC ATT ATA 3456 Ser Val Phe Leu Asn Tyr Lys Leu Tyr Glu GlyVal Glu Val Ile Ile 1140 1145 1150 AGA AAA AAT GGT CCT ATA GAT ATA TCTAAT ACA GAT AAT TTT GTT AGA 3504 Arg Lys Asn Gly Pro Ile Asp Ile Ser AsnThr Asp Asn Phe Val Arg 1155 1160 1165 AAA AAC GAT CTA GCA TAC ATT AATGTA GTA GAT CGT GGT GTA GAA TAT 3552 Lys Asn Asp Leu Ala Tyr Ile Asn ValVal Asp Arg Gly Val Glu Tyr 1170 1175 1180 CGG TTA TAT GCT GAT ACA AAATCA GAG AAA GAG AAA ATA ATA AGA ACA 3600 Arg Leu Tyr Ala Asp Thr Lys SerGlu Lys Glu Lys Ile Ile Arg Thr 1185 1190 1195 1200 TCT AAT CTA AAC GATAGC TTA GGT CAA ATT ATA GTT ATG GAT TCA ATA 3648 Ser Asn Leu Asn Asp SerLeu Gly Gln Ile Ile Val Met Asp Ser Ile 1205 1210 1215 GGA AAT AAT TGCACA ATG AAT TTT CAA AAC AAT AAT GGG AGC AAT ATA 3696 Gly Asn Asn Cys ThrMet Asn Phe Gln Asn Asn Asn Gly Ser Asn Ile 1220 1225 1230 GGA TTA CTAGGT TTT CAT TCA AAT AAT TTG GTT GCT AGT AGT TGG TAT 3744 Gly Leu Leu GlyPhe His Ser Asn Asn Leu Val Ala Ser Ser Trp Tyr 1235 1240 1245 TAT AACAAT ATA CGA AGA AAT ACT AGC AGT AAT GGA TGC TTT TGG AGT 3792 Tyr Asn AsnIle Arg Arg Asn Thr Ser Ser Asn Gly Cys Phe Trp Ser 1250 1255 1260 TCTATT TCT AAA GAG AAT GGA TGG AAA GAA TGA 3825 Ser Ile Ser Lys Glu Asn GlyTrp Lys Glu 1265 1270 1274 amino acids amino acid linear protein 71 MetPro Val Ala Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp 1 5 10 15Asp Thr Ile Leu Tyr Met Gln Ile Pro Tyr Glu Glu Lys Ser Lys Lys 20 25 30Tyr Tyr Lys Ala Phe Glu Ile Met Arg Asn Val Trp Ile Ile Pro Glu 35 40 45Arg Asn Thr Ile Gly Thr Asn Pro Ser Asp Phe Asp Pro Pro Ala Ser 50 55 60Leu Lys Asn Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Leu Thr Thr 65 70 7580 Asp Ala Glu Lys Asp Arg Tyr Leu Lys Thr Thr Ile Lys Leu Phe Lys 85 9095 Arg Ile Asn Ser Asn Pro Ala Gly Lys Val Leu Leu Gln Glu Ile Ser 100105 110 Tyr Ala Lys Pro Tyr Leu Gly Asn Asp His Thr Pro Ile Asp Glu Phe115 120 125 Ser Pro Val Thr Arg Thr Thr Ser Val Asn Ile Lys Leu Ser ThrAsn 130 135 140 Val Glu Ser Ser Met Leu Leu Asn Leu Leu Val Leu Gly AlaGly Pro 145 150 155 160 Asp Ile Phe Glu Ser Cys Cys Tyr Pro Val Arg LysLeu Ile Asp Pro 165 170 175 Asp Val Val Tyr Asp Pro Ser Asn Tyr Gly PheGly Ser Ile Asn Ile 180 185 190 Val Thr Phe Ser Pro Glu Tyr Glu Tyr ThrPhe Asn Asp Ile Ser Gly 195 200 205 Gly His Asn Ser Ser Thr Glu Ser PheIle Ala Asp Pro Ala Ile Ser 210 215 220 Leu Ala His Glu Leu Ile His AlaLeu His Gly Leu Tyr Gly Ala Arg 225 230 235 240 Gly Val Thr Tyr Glu GluThr Ile Glu Val Lys Gln Ala Pro Leu Met 245 250 255 Ile Ala Glu Lys ProIle Arg Leu Glu Glu Phe Leu Thr Phe Gly Gly 260 265 270 Gln Asp Leu AsnIle Ile Thr Ser Ala Met Lys Glu Lys Ile Tyr Asn 275 280 285 Asn Leu LeuAla Asn Tyr Glu Lys Ile Ala Thr Arg Leu Ser Glu Val 290 295 300 Asn SerAla Pro Pro Glu Tyr Asp Ile Asn Glu Tyr Lys Asp Tyr Phe 305 310 315 320Gln Trp Lys Tyr Gly Leu Asp Lys Asn Ala Asp Gly Ser Tyr Thr Val 325 330335 Asn Glu Asn Lys Phe Asn Glu Ile Tyr Lys Lys Leu Tyr Ser Phe Thr 340345 350 Glu Ser Asp Leu Ala Asn Lys Phe Lys Val Lys Cys Arg Asn Thr Tyr355 360 365 Phe Ile Lys Tyr Glu Phe Leu Lys Val Pro Asn Leu Leu Asp AspAsp 370 375 380 Ile Tyr Thr Val Ser Glu Gly Phe Asn Ile Gly Asn Leu AlaVal Asn 385 390 395 400 Asn Arg Gly Gln Ser Ile Lys Leu Asn Pro Lys IleIle Asp Ser Ile 405 410 415 Pro Asp Lys Gly Leu Val Glu Lys Ile Val LysPhe Cys Lys Ser Val 420 425 430 Ile Pro Arg Lys Gly Thr Lys Ala Pro ProArg Leu Cys Ile Arg Val 435 440 445 Asn Asn Ser Glu Leu Phe Phe Val AlaSer Glu Ser Ser Tyr Asn Glu 450 455 460 Asn Asp Ile Asn Thr Pro Lys GluIle Asp Asp Thr Thr Asn Leu Asn 465 470 475 480 Asn Asn Tyr Arg Asn AsnLeu Asp Glu Val Ile Leu Asp Tyr Asn Ser 485 490 495 Gln Thr Ile Pro GlnIle Ser Asn Arg Thr Leu Asn Thr Leu Val Gln 500 505 510 Asp Asn Ser TyrVal Pro Arg Tyr Asp Ser Asn Gly Thr Ser Glu Ile 515 520 525 Glu Glu TyrAsp Val Val Asp Phe Asn Val Phe Phe Tyr Leu His Ala 530 535 540 Gln LysVal Pro Glu Gly Glu Thr Asn Ile Ser Leu Thr Ser Ser Ile 545 550 555 560Asp Thr Ala Leu Leu Glu Glu Ser Lys Asp Ile Phe Phe Ser Ser Glu 565 570575 Phe Ile Asp Thr Ile Asn Lys Pro Val Asn Ala Ala Leu Phe Ile Asp 580585 590 Trp Ile Ser Lys Val Ile Arg Asp Phe Thr Thr Glu Ala Thr Gln Lys595 600 605 Ser Thr Val Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro TyrVal 610 615 620 Gly Leu Ala Leu Asn Ile Ile Ile Glu Ala Glu Lys Gly AsnPhe Glu 625 630 635 640 Glu Ala Phe Glu Leu Leu Gly Val Gly Ile Leu LeuGlu Phe Val Pro 645 650 655 Glu Leu Thr Ile Pro Val Ile Leu Val Phe ThrIle Lys Ser Tyr Ile 660 665 670 Asp Ser Tyr Glu Asn Lys Asn Lys Ala IleLys Ala Ile Asn Asn Ser 675 680 685 Leu Ile Glu Arg Glu Ala Lys Trp LysGlu Ile Tyr Ser Trp Ile Val 690 695 700 Ser Asn Trp Leu Thr Arg Ile AsnThr Gln Phe Asn Lys Arg Lys Glu 705 710 715 720 Gln Met Tyr Gln Ala LeuGln Asn Gln Val Asp Ala Ile Lys Thr Ala 725 730 735 Ile Glu Tyr Lys TyrAsn Asn Tyr Thr Ser Asp Glu Lys Asn Arg Leu 740 745 750 Glu Ser Glu TyrAsn Ile Asn Asn Ile Glu Glu Glu Leu Asn Lys Lys 755 760 765 Val Ser LeuAla Met Lys Asn Ile Glu Arg Phe Met Thr Glu Ser Ser 770 775 780 Ile SerTyr Leu Met Lys Leu Ile Asn Glu Ala Lys Val Gly Lys Leu 785 790 795 800Lys Lys Tyr Asp Asn His Val Lys Ser Asp Leu Leu Asn Tyr Ile Leu 805 810815 Asp His Arg Ser Ile Leu Gly Glu Gln Thr Asn Glu Leu Ser Asp Leu 820825 830 Val Thr Ser Thr Leu Asn Ser Ser Ile Pro Phe Glu Leu Ser Ser Tyr835 840 845 Thr Asn Asp Lys Ile Leu Ile Ile Tyr Phe Asn Arg Leu Tyr LysLys 850 855 860 Ile Lys Asp Ser Ser Ile Leu Asp Met Arg Tyr Glu Asn AsnLys Phe 865 870 875 880 Ile Asp Ile Ser Gly Tyr Gly Ser Asn Ile Ser IleAsn Gly Asn Val 885 890 895 Tyr Ile Tyr Ser Thr Asn Arg Asn Gln Phe GlyIle Tyr Asn Ser Arg 900 905 910 Leu Ser Glu Val Asn Ile Ala Gln Asn AsnAsp Ile Ile Tyr Asn Ser 915 920 925 Arg Tyr Gln Asn Phe Ser Ile Ser PheTrp Val Arg Ile Pro Lys His 930 935 940 Tyr Lys Pro Met Asn His Asn ArgGlu Tyr Thr Ile Ile Asn Cys Met 945 950 955 960 Gly Asn Asn Asn Ser GlyTrp Lys Ile Ser Leu Arg Thr Val Arg Asp 965 970 975 Cys Glu Ile Ile TrpThr Leu Gln Asp Thr Ser Gly Asn Lys Glu Asn 980 985 990 Leu Ile Phe ArgTyr Glu Glu Leu Asn Arg Ile Ser Asn Tyr Ile Asn 995 1000 1005 Lys TrpIle Phe Val Thr Ile Thr Asn Asn Arg Leu Gly Asn Ser Arg 1010 1015 1020Ile Tyr Ile Asn Gly Asn Leu Ile Val Glu Lys Ser Ile Ser Asn Leu 10251030 1035 1040 Gly Asp Ile His Val Ser Asp Asn Ile Leu Phe Lys Ile ValGly Cys 1045 1050 1055 Asp Asp Glu Thr Tyr Val Gly Ile Arg Tyr Phe LysVal Phe Asn Thr 1060 1065 1070 Glu Leu Asp Lys Thr Glu Ile Glu Thr LeuTyr Ser Asn Glu Pro Asp 1075 1080 1085 Pro Ser Ile Leu Lys Asn Tyr TrpGly Asn Tyr Leu Leu Tyr Asn Lys 1090 1095 1100 Lys Tyr Tyr Leu Phe AsnLeu Leu Arg Lys Asp Lys Tyr Ile Thr Leu 1105 1110 1115 1120 Asn Ser GlyIle Leu Asn Ile Asn Gln Gln Arg Gly Val Thr Glu Gly 1125 1130 1135 SerVal Phe Leu Asn Tyr Lys Leu Tyr Glu Gly Val Glu Val Ile Ile 1140 11451150 Arg Lys Asn Gly Pro Ile Asp Ile Ser Asn Thr Asp Asn Phe Val Arg1155 1160 1165 Lys Asn Asp Leu Ala Tyr Ile Asn Val Val Asp Arg Gly ValGlu Tyr 1170 1175 1180 Arg Leu Tyr Ala Asp Thr Lys Ser Glu Lys Glu LysIle Ile Arg Thr 1185 1190 1195 1200 Ser Asn Leu Asn Asp Ser Leu Gly GlnIle Ile Val Met Asp Ser Ile 1205 1210 1215 Gly Asn Asn Cys Thr Met AsnPhe Gln Asn Asn Asn Gly Ser Asn Ile 1220 1225 1230 Gly Leu Leu Gly PheHis Ser Asn Asn Leu Val Ala Ser Ser Trp Tyr 1235 1240 1245 Tyr Asn AsnIle Arg Arg Asn Thr Ser Ser Asn Gly Cys Phe Trp Ser 1250 1255 1260 SerIle Ser Lys Glu Asn Gly Trp Lys Glu 1265 1270 1460 base pairs nucleicacid double linear DNA (genomic) CDS 108..1451 72 AGATCTCGAT CCCGCGAAATTAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60 TTCCCCTCTA GAAATAATTTTGTTTAACTT TAAGAAGGAG ATATACC ATG GGC CAT 116 Met Gly His 1 CAT CAT CATCAT CAT CAT CAT CAT CAC AGC AGC GGC CAT ATC GAA GGT 164 His His His HisHis His His His His Ser Ser Gly His Ile Glu Gly 5 10 15 CGT CAT ATG GCTAGC ATG GCT ATT CTA ATT ATA TAT TTT AAT AGA TTA 212 Arg His Met Ala SerMet Ala Ile Leu Ile Ile Tyr Phe Asn Arg Leu 20 25 30 35 TAT AAA AAA ATTAAA GAT AGT TCT ATT TTA GAT ATG CGA TAT GAA AAT 260 Tyr Lys Lys Ile LysAsp Ser Ser Ile Leu Asp Met Arg Tyr Glu Asn 40 45 50 AAT AAA TTT ATA GATATC TCT GGA TAT GGT TCA AAT ATA AGC ATT AAT 308 Asn Lys Phe Ile Asp IleSer Gly Tyr Gly Ser Asn Ile Ser Ile Asn 55 60 65 GGA AAC GTA TAT ATT TATTCA ACA AAT AGA AAT CAA TTT GGA ATA TAT 356 Gly Asn Val Tyr Ile Tyr SerThr Asn Arg Asn Gln Phe Gly Ile Tyr 70 75 80 AAT AGT AGG CTT AGT GAA GTTAAT ATA GCT CAA AAT AAT GAT ATT ATA 404 Asn Ser Arg Leu Ser Glu Val AsnIle Ala Gln Asn Asn Asp Ile Ile 85 90 95 TAC AAT AGT AGA TAT CAA AAT TTTAGT ATT AGT TTC TGG GTA AGG ATT 452 Tyr Asn Ser Arg Tyr Gln Asn Phe SerIle Ser Phe Trp Val Arg Ile 100 105 110 115 CCT AAA CAC TAC AAA CCT ATGAAT CAT AAT CGG GAA TAC ACT ATA ATA 500 Pro Lys His Tyr Lys Pro Met AsnHis Asn Arg Glu Tyr Thr Ile Ile 120 125 130 AAT TGT ATG GGG AAT AAT AATTCG GGA TGG AAA ATA TCA CTT AGA ACT 548 Asn Cys Met Gly Asn Asn Asn SerGly Trp Lys Ile Ser Leu Arg Thr 135 140 145 GTT AGA GAT TGT GAA ATA ATTTGG ACT TTA CAA GAT ACT TCT GGA AAT 596 Val Arg Asp Cys Glu Ile Ile TrpThr Leu Gln Asp Thr Ser Gly Asn 150 155 160 AAG GAA AAT TTA ATT TTT AGGTAT GAA GAA CTT AAT AGG ATA TCT AAT 644 Lys Glu Asn Leu Ile Phe Arg TyrGlu Glu Leu Asn Arg Ile Ser Asn 165 170 175 TAT ATA AAT AAA TGG ATT TTTGTA ACT ATT ACT AAT AAT AGA TTA GGC 692 Tyr Ile Asn Lys Trp Ile Phe ValThr Ile Thr Asn Asn Arg Leu Gly 180 185 190 195 AAT TCT AGA ATT TAC ATCAAT GGA AAT TTA ATA GTT GAA AAA TCA ATT 740 Asn Ser Arg Ile Tyr Ile AsnGly Asn Leu Ile Val Glu Lys Ser Ile 200 205 210 TCG AAT TTA GGT GAT ATTCAT GTT AGT GAT AAT ATA TTA TTT AAA ATT 788 Ser Asn Leu Gly Asp Ile HisVal Ser Asp Asn Ile Leu Phe Lys Ile 215 220 225 GTT GGT TGT GAT GAT GAAACG TAT GTT GGT ATA AGA TAT TTT AAA GTT 836 Val Gly Cys Asp Asp Glu ThrTyr Val Gly Ile Arg Tyr Phe Lys Val 230 235 240 TTT AAT ACG GAA TTA GATAAA ACA GAA ATT GAG ACT TTA TAT AGT AAT 884 Phe Asn Thr Glu Leu Asp LysThr Glu Ile Glu Thr Leu Tyr Ser Asn 245 250 255 GAG CCA GAT CCA AGT ATCTTA AAA AAC TAT TGG GGA AAT TAT TTG CTA 932 Glu Pro Asp Pro Ser Ile LeuLys Asn Tyr Trp Gly Asn Tyr Leu Leu 260 265 270 275 TAT AAT AAA AAA TATTAT TTA TTC AAT TTA CTA AGA AAA GAT AAG TAT 980 Tyr Asn Lys Lys Tyr TyrLeu Phe Asn Leu Leu Arg Lys Asp Lys Tyr 280 285 290 ATT ACT CTG AAT TCAGGC ATT TTA AAT ATT AAT CAA CAA AGA GGT GTT 1028 Ile Thr Leu Asn Ser GlyIle Leu Asn Ile Asn Gln Gln Arg Gly Val 295 300 305 ACT GAA GGC TCT GTTTTT TTG AAC TAT AAA TTA TAT GAA GGA GTA GAA 1076 Thr Glu Gly Ser Val PheLeu Asn Tyr Lys Leu Tyr Glu Gly Val Glu 310 315 320 GTC ATT ATA AGA AAAAAT GGT CCT ATA GAT ATA TCT AAT ACA GAT AAT 1124 Val Ile Ile Arg Lys AsnGly Pro Ile Asp Ile Ser Asn Thr Asp Asn 325 330 335 TTT GTT AGA AAA AACGAT CTA GCA TAC ATT AAT GTA GTA GAT CGT GGT 1172 Phe Val Arg Lys Asn AspLeu Ala Tyr Ile Asn Val Val Asp Arg Gly 340 345 350 355 GTA GAA TAT CGGTTA TAT GCT GAT ACA AAA TCA GAG AAA GAG AAA ATA 1220 Val Glu Tyr Arg LeuTyr Ala Asp Thr Lys Ser Glu Lys Glu Lys Ile 360 365 370 ATA AGA ACA TCTAAT CTA AAC GAT AGC TTA GGT CAA ATT ATA GTT ATG 1268 Ile Arg Thr Ser AsnLeu Asn Asp Ser Leu Gly Gln Ile Ile Val Met 375 380 385 GAT TCA ATA GGAAAT AAT TGC ACA ATG AAT TTT CAA AAC AAT AAT GGG 1316 Asp Ser Ile Gly AsnAsn Cys Thr Met Asn Phe Gln Asn Asn Asn Gly 390 395 400 AGC AAT ATA GGATTA CTA GGT TTT CAT TCA AAT AAT TTG GTT GCT AGT 1364 Ser Asn Ile Gly LeuLeu Gly Phe His Ser Asn Asn Leu Val Ala Ser 405 410 415 AGT TGG TAT TATAAC AAT ATA CGA AGA AAT ACT AGC AGT AAT GGA TGC 1412 Ser Trp Tyr Tyr AsnAsn Ile Arg Arg Asn Thr Ser Ser Asn Gly Cys 420 425 430 435 TTT TGG AGTTCT ATT TCT AAA GAG AAT GGA TGG AAA GAA TGAAAGCTT 1460 Phe Trp Ser SerIle Ser Lys Glu Asn Gly Trp Lys Glu 440 445 448 amino acids amino acidlinear protein 73 Met Gly His His His His His His His His His His SerSer Gly His 1 5 10 15 Ile Glu Gly Arg His Met Ala Ser Met Ala Ile LeuIle Ile Tyr Phe 20 25 30 Asn Arg Leu Tyr Lys Lys Ile Lys Asp Ser Ser IleLeu Asp Met Arg 35 40 45 Tyr Glu Asn Asn Lys Phe Ile Asp Ile Ser Gly TyrGly Ser Asn Ile 50 55 60 Ser Ile Asn Gly Asn Val Tyr Ile Tyr Ser Thr AsnArg Asn Gln Phe 65 70 75 80 Gly Ile Tyr Asn Ser Arg Leu Ser Glu Val AsnIle Ala Gln Asn Asn 85 90 95 Asp Ile Ile Tyr Asn Ser Arg Tyr Gln Asn PheSer Ile Ser Phe Trp 100 105 110 Val Arg Ile Pro Lys His Tyr Lys Pro MetAsn His Asn Arg Glu Tyr 115 120 125 Thr Ile Ile Asn Cys Met Gly Asn AsnAsn Ser Gly Trp Lys Ile Ser 130 135 140 Leu Arg Thr Val Arg Asp Cys GluIle Ile Trp Thr Leu Gln Asp Thr 145 150 155 160 Ser Gly Asn Lys Glu AsnLeu Ile Phe Arg Tyr Glu Glu Leu Asn Arg 165 170 175 Ile Ser Asn Tyr IleAsn Lys Trp Ile Phe Val Thr Ile Thr Asn Asn 180 185 190 Arg Leu Gly AsnSer Arg Ile Tyr Ile Asn Gly Asn Leu Ile Val Glu 195 200 205 Lys Ser IleSer Asn Leu Gly Asp Ile His Val Ser Asp Asn Ile Leu 210 215 220 Phe LysIle Val Gly Cys Asp Asp Glu Thr Tyr Val Gly Ile Arg Tyr 225 230 235 240Phe Lys Val Phe Asn Thr Glu Leu Asp Lys Thr Glu Ile Glu Thr Leu 245 250255 Tyr Ser Asn Glu Pro Asp Pro Ser Ile Leu Lys Asn Tyr Trp Gly Asn 260265 270 Tyr Leu Leu Tyr Asn Lys Lys Tyr Tyr Leu Phe Asn Leu Leu Arg Lys275 280 285 Asp Lys Tyr Ile Thr Leu Asn Ser Gly Ile Leu Asn Ile Asn GlnGln 290 295 300 Arg Gly Val Thr Glu Gly Ser Val Phe Leu Asn Tyr Lys LeuTyr Glu 305 310 315 320 Gly Val Glu Val Ile Ile Arg Lys Asn Gly Pro IleAsp Ile Ser Asn 325 330 335 Thr Asp Asn Phe Val Arg Lys Asn Asp Leu AlaTyr Ile Asn Val Val 340 345 350 Asp Arg Gly Val Glu Tyr Arg Leu Tyr AlaAsp Thr Lys Ser Glu Lys 355 360 365 Glu Lys Ile Ile Arg Thr Ser Asn LeuAsn Asp Ser Leu Gly Gln Ile 370 375 380 Ile Val Met Asp Ser Ile Gly AsnAsn Cys Thr Met Asn Phe Gln Asn 385 390 395 400 Asn Asn Gly Ser Asn IleGly Leu Leu Gly Phe His Ser Asn Asn Leu 405 410 415 Val Ala Ser Ser TrpTyr Tyr Asn Asn Ile Arg Arg Asn Thr Ser Ser 420 425 430 Asn Gly Cys PheTrp Ser Ser Ile Ser Lys Glu Asn Gly Trp Lys Glu 435 440 445 33 basepairs nucleic acid single linear other nucleic acid /desc = “DNA” 74CGCCATGGCT ATTCTAATTA TATATTTTAA TAG 33 29 base pairs nucleic acidsingle linear other nucleic acid /desc = “DNA” 75 GCAAGCTTTC ATTCTTTCCATCCATTCTC 29 3894 base pairs nucleic acid double linear DNA (genomic)CDS 1..3891 76 ATG CCA GTT AAT ATA AAA AAC TTT AAT TAT AAT GAC CCT ATTAAT AAT 48 Met Pro Val Asn Ile Lys Asn Phe Asn Tyr Asn Asp Pro Ile AsnAsn 1 5 10 15 GAT GAC ATT ATT ATG ATG GAA CCA TTC AAT GAC CCA GGG CCAGGA ACA 96 Asp Asp Ile Ile Met Met Glu Pro Phe Asn Asp Pro Gly Pro GlyThr 20 25 30 TAT TAT AAA GCT TTT AGG ATT ATA GAT CGT ATT TGG ATA GTA CCAGAA 144 Tyr Tyr Lys Ala Phe Arg Ile Ile Asp Arg Ile Trp Ile Val Pro Glu35 40 45 AGG TTT ACT TAT GGA TTT CAA CCT GAC CAA TTT AAT GCC AGT ACA GGA192 Arg Phe Thr Tyr Gly Phe Gln Pro Asp Gln Phe Asn Ala Ser Thr Gly 5055 60 GTT TTT AGT AAA GAT GTC TAC GAA TAT TAC GAT CCA ACT TAT TTA AAA240 Val Phe Ser Lys Asp Val Tyr Glu Tyr Tyr Asp Pro Thr Tyr Leu Lys 6570 75 80 ACC GAT GCT GAA AAA GAT AAA TTT TTA AAA ACA ATG ATT AAA TTA TTT288 Thr Asp Ala Glu Lys Asp Lys Phe Leu Lys Thr Met Ile Lys Leu Phe 8590 95 AAT AGA ATT AAT TCA AAA CCA TCA GGA CAG AGA TTA CTG GAT ATG ATA336 Asn Arg Ile Asn Ser Lys Pro Ser Gly Gln Arg Leu Leu Asp Met Ile 100105 110 GTA GAT GCT ATA CCT TAT CTT GGA AAT GCA TCT ACA CCG CCC GAC AAA384 Val Asp Ala Ile Pro Tyr Leu Gly Asn Ala Ser Thr Pro Pro Asp Lys 115120 125 TTT GCA GCA AAT GTT GCA AAT GTA TCT ATT AAT AAA AAA ATT ATC CAA432 Phe Ala Ala Asn Val Ala Asn Val Ser Ile Asn Lys Lys Ile Ile Gln 130135 140 CCT GGA GCT GAA GAT CAA ATA AAA GGT TTA ATG ACA AAT TTA ATA ATA480 Pro Gly Ala Glu Asp Gln Ile Lys Gly Leu Met Thr Asn Leu Ile Ile 145150 155 160 TTT GGA CCA GGA CCA GTT CTA AGT GAT AAT TTT ACT GAT AGT ATGATT 528 Phe Gly Pro Gly Pro Val Leu Ser Asp Asn Phe Thr Asp Ser Met Ile165 170 175 ATG AAT GGC CAT TCC CCA ATA TCA GAA GGA TTT GGT GCA AGA ATGATG 576 Met Asn Gly His Ser Pro Ile Ser Glu Gly Phe Gly Ala Arg Met Met180 185 190 ATA AGA TTT TGT CCT AGT TGT TTA AAT GTA TTT AAT AAT GTT CAGGAA 624 Ile Arg Phe Cys Pro Ser Cys Leu Asn Val Phe Asn Asn Val Gln Glu195 200 205 AAT AAA GAT ACA TCT ATA TTT AGT AGA CGC GCG TAT TTT GCA GATCCA 672 Asn Lys Asp Thr Ser Ile Phe Ser Arg Arg Ala Tyr Phe Ala Asp Pro210 215 220 GCT CTA ACG TTA ATG CAT GAA CTT ATA CAT GTG TTA CAT GGA TTATAT 720 Ala Leu Thr Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr225 230 235 240 GGA ATT AAG ATA AGT AAT TTA CCA ATT ACT CCA AAT ACA AAAGAA TTT 768 Gly Ile Lys Ile Ser Asn Leu Pro Ile Thr Pro Asn Thr Lys GluPhe 245 250 255 TTC ATG CAA CAT AGC GAT CCT GTA CAA GCA GAA GAA CTA TATACA TTC 816 Phe Met Gln His Ser Asp Pro Val Gln Ala Glu Glu Leu Tyr ThrPhe 260 265 270 GGA GGA CAT GAT CCT AGT GTT ATA AGT CCT TCT ACG GAT ATGAAT ATT 864 Gly Gly His Asp Pro Ser Val Ile Ser Pro Ser Thr Asp Met AsnIle 275 280 285 TAT AAT AAA GCG TTA CAA AAT TTT CAA GAT ATA GCT AAT AGGCTT AAT 912 Tyr Asn Lys Ala Leu Gln Asn Phe Gln Asp Ile Ala Asn Arg LeuAsn 290 295 300 ATT GTT TCA AGT GCC CAA GGG AGT GGA ATT GAT ATT TCC TTATAT AAA 960 Ile Val Ser Ser Ala Gln Gly Ser Gly Ile Asp Ile Ser Leu TyrLys 305 310 315 320 CAA ATA TAT AAA AAT AAA TAT GAT TTT GTT GAA GAT CCTAAT GGA AAA 1008 Gln Ile Tyr Lys Asn Lys Tyr Asp Phe Val Glu Asp Pro AsnGly Lys 325 330 335 TAT AGT GTA GAT AAG GAT AAG TTT GAT AAA TTA TAT AAGGCC TTA ATG 1056 Tyr Ser Val Asp Lys Asp Lys Phe Asp Lys Leu Tyr Lys AlaLeu Met 340 345 350 TTT GGC TTT ACT GAA ACT AAT CTA GCT GGT GAA TAT GGAATA AAA ACT 1104 Phe Gly Phe Thr Glu Thr Asn Leu Ala Gly Glu Tyr Gly IleLys Thr 355 360 365 AGG TAT TCT TAT TTT AGT GAA TAT TTG CCA CCG ATA AAAACT GAA AAA 1152 Arg Tyr Ser Tyr Phe Ser Glu Tyr Leu Pro Pro Ile Lys ThrGlu Lys 370 375 380 TTG TTA GAC AAT ACA ATT TAT ACT CAA AAT GAA GGC TTTAAC ATA GCT 1200 Leu Leu Asp Asn Thr Ile Tyr Thr Gln Asn Glu Gly Phe AsnIle Ala 385 390 395 400 AGT AAA AAT CTC AAA ACG GAA TTT AAT GGT CAG AATAAG GCG GTA AAT 1248 Ser Lys Asn Leu Lys Thr Glu Phe Asn Gly Gln Asn LysAla Val Asn 405 410 415 AAA GAG GCT TAT GAA GAA ATC AGC CTA GAA CAT CTCGTT ATA TAT AGA 1296 Lys Glu Ala Tyr Glu Glu Ile Ser Leu Glu His Leu ValIle Tyr Arg 420 425 430 ATA GCA ATG TGC AAG CCT GTA ATG TAC AAA AAT ACCGGT AAA TCT GAA 1344 Ile Ala Met Cys Lys Pro Val Met Tyr Lys Asn Thr GlyLys Ser Glu 435 440 445 CAG TGT ATT ATT GTT AAT AAT GAG GAT TTA TTT TTCATA GCT AAT AAA 1392 Gln Cys Ile Ile Val Asn Asn Glu Asp Leu Phe Phe IleAla Asn Lys 450 455 460 GAT AGT TTT TCA AAA GAT TTA GCT AAA GCA GAA ACTATA GCA TAT AAT 1440 Asp Ser Phe Ser Lys Asp Leu Ala Lys Ala Glu Thr IleAla Tyr Asn 465 470 475 480 ACA CAA AAT AAT ACT ATA GAA AAT AAT TTT TCTATA GAT CAG TTG ATT 1488 Thr Gln Asn Asn Thr Ile Glu Asn Asn Phe Ser IleAsp Gln Leu Ile 485 490 495 TTA GAT AAT GAT TTA AGC AGT GGC ATA GAC TTACCA AAT GAA AAC ACA 1536 Leu Asp Asn Asp Leu Ser Ser Gly Ile Asp Leu ProAsn Glu Asn Thr 500 505 510 GAA CCA TTT ACA AAT TTT GAC GAC ATA GAT ATCCCT GTG TAT ATT AAA 1584 Glu Pro Phe Thr Asn Phe Asp Asp Ile Asp Ile ProVal Tyr Ile Lys 515 520 525 CAA TCT GCT TTA AAA AAA ATT TTT GTG GAT GGAGAT AGC CTT TTT GAA 1632 Gln Ser Ala Leu Lys Lys Ile Phe Val Asp Gly AspSer Leu Phe Glu 530 535 540 TAT TTA CAT GCT CAA ACA TTT CCT TCT AAT ATAGAA AAT CTA CAA CTA 1680 Tyr Leu His Ala Gln Thr Phe Pro Ser Asn Ile GluAsn Leu Gln Leu 545 550 555 560 ACG AAT TCA TTA AAT GAT GCT TTA AGA AATAAT AAT AAA GTC TAT ACT 1728 Thr Asn Ser Leu Asn Asp Ala Leu Arg Asn AsnAsn Lys Val Tyr Thr 565 570 575 TTT TTT TCT ACA AAC CTT GTT GAA AAA GCTAAT ACA GTT GTA GGT GCT 1776 Phe Phe Ser Thr Asn Leu Val Glu Lys Ala AsnThr Val Val Gly Ala 580 585 590 TCA CTT TTT GTA AAC TGG GTA AAA GGA GTAATA GAT GAT TTT ACA TCT 1824 Ser Leu Phe Val Asn Trp Val Lys Gly Val IleAsp Asp Phe Thr Ser 595 600 605 GAA TCC ACA CAA AAA AGT ACT ATA GAT AAAGTT TCA GAT GTA TCC ATA 1872 Glu Ser Thr Gln Lys Ser Thr Ile Asp Lys ValSer Asp Val Ser Ile 610 615 620 ATT ATT CCC TAT ATA GGA CCT GCT TTG AATGTA GGA AAT GAA ACA GCT 1920 Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn ValGly Asn Glu Thr Ala 625 630 635 640 AAA GAA AAT TTT AAA AAT GCT TTT GAAATA GGT GGA GCC GCT ATC TTA 1968 Lys Glu Asn Phe Lys Asn Ala Phe Glu IleGly Gly Ala Ala Ile Leu 645 650 655 ATG GAG TTT ATT CCA GAA CTT ATT GTACCT ATA GTT GGA TTT TTT ACA 2016 Met Glu Phe Ile Pro Glu Leu Ile Val ProIle Val Gly Phe Phe Thr 660 665 670 TTA GAA TCA TAT GTA GGA AAT AAA GGGCAT ATT ATT ATG ACG ATA TCC 2064 Leu Glu Ser Tyr Val Gly Asn Lys Gly HisIle Ile Met Thr Ile Ser 675 680 685 AAT GCT TTA AAG AAA AGG GAT CAA AAATGG ACA GAT ATG TAT GGT TTG 2112 Asn Ala Leu Lys Lys Arg Asp Gln Lys TrpThr Asp Met Tyr Gly Leu 690 695 700 ATA GTA TCG CAG TGG CTC TCA ACG GTTAAT ACT CAA TTT TAT ACA ATA 2160 Ile Val Ser Gln Trp Leu Ser Thr Val AsnThr Gln Phe Tyr Thr Ile 705 710 715 720 AAA GAA AGA ATG TAC AAT GCT TTAAAT AAT CAA TCA CAA GCA ATA GAA 2208 Lys Glu Arg Met Tyr Asn Ala Leu AsnAsn Gln Ser Gln Ala Ile Glu 725 730 735 AAA ATA ATA GAA GAT CAA TAT AATAGA TAT AGT GAA GAA GAT AAA ATG 2256 Lys Ile Ile Glu Asp Gln Tyr Asn ArgTyr Ser Glu Glu Asp Lys Met 740 745 750 AAT ATT AAC ATT GAT TTT AAT GATATA GAT TTT AAA CTT AAT CAA AGT 2304 Asn Ile Asn Ile Asp Phe Asn Asp IleAsp Phe Lys Leu Asn Gln Ser 755 760 765 ATA AAT TTA GCA ATA AAC AAT ATAGAT GAT TTT ATA AAC CAA TGT TCT 2352 Ile Asn Leu Ala Ile Asn Asn Ile AspAsp Phe Ile Asn Gln Cys Ser 770 775 780 ATA TCA TAT CTA ATG AAT AGA ATGATT CCA TTA GCT GTA AAA AAG TTA 2400 Ile Ser Tyr Leu Met Asn Arg Met IlePro Leu Ala Val Lys Lys Leu 785 790 795 800 AAA GAC TTT GAT GAT AAT CTTAAG AGA GAT TTA TTG GAG TAT ATA GAT 2448 Lys Asp Phe Asp Asp Asn Leu LysArg Asp Leu Leu Glu Tyr Ile Asp 805 810 815 ACA AAT GAA CTA TAT TTA CTTGAT GAA GTA AAT ATT CTA AAA TCA AAA 2496 Thr Asn Glu Leu Tyr Leu Leu AspGlu Val Asn Ile Leu Lys Ser Lys 820 825 830 GTA AAT AGA CAC CTA AAA GACAGT ATA CCA TTT GAT CTT TCA CTA TAT 2544 Val Asn Arg His Leu Lys Asp SerIle Pro Phe Asp Leu Ser Leu Tyr 835 840 845 ACC AAG GAC ACA ATT TTA ATACAA GTT TTT AAT AAT TAT ATT AGT AAT 2592 Thr Lys Asp Thr Ile Leu Ile GlnVal Phe Asn Asn Tyr Ile Ser Asn 850 855 860 ATT AGT AGT AAT GCT ATT TTAAGT TTA AGT TAT AGA GGT GGG CGT TTA 2640 Ile Ser Ser Asn Ala Ile Leu SerLeu Ser Tyr Arg Gly Gly Arg Leu 865 870 875 880 ATA GAT TCA TCT GGA TATGGT GCA ACT ATG AAT GTA GGT TCA GAT GTT 2688 Ile Asp Ser Ser Gly Tyr GlyAla Thr Met Asn Val Gly Ser Asp Val 885 890 895 ATC TTT AAT GAT ATA GGAAAT GGT CAA TTT AAA TTA AAT AAT TCT GAA 2736 Ile Phe Asn Asp Ile Gly AsnGly Gln Phe Lys Leu Asn Asn Ser Glu 900 905 910 AAT AGT AAT ATT ACG GCACAT CAA AGT AAA TTC GTT GTA TAT GAT AGT 2784 Asn Ser Asn Ile Thr Ala HisGln Ser Lys Phe Val Val Tyr Asp Ser 915 920 925 ATG TTT GAT AAT TTT AGCATT AAC TTT TGG GTA AGG ACT CCT AAA TAT 2832 Met Phe Asp Asn Phe Ser IleAsn Phe Trp Val Arg Thr Pro Lys Tyr 930 935 940 AAT AAT AAT GAT ATA CAAACT TAT CTT CAA AAT GAG TAT ACA ATA ATT 2880 Asn Asn Asn Asp Ile Gln ThrTyr Leu Gln Asn Glu Tyr Thr Ile Ile 945 950 955 960 AGT TGT ATA AAA AATGAC TCA GGA TGG AAA GTA TCT ATT AAG GGA AAT 2928 Ser Cys Ile Lys Asn AspSer Gly Trp Lys Val Ser Ile Lys Gly Asn 965 970 975 AGA ATA ATA TGG ACATTA ATA GAT GTT AAT GCA AAA TCT AAA TCA ATA 2976 Arg Ile Ile Trp Thr LeuIle Asp Val Asn Ala Lys Ser Lys Ser Ile 980 985 990 TTT TTC GAA TAT AGTATA AAA GAT AAT ATA TCA GAT TAT ATA AAT AAA 3024 Phe Phe Glu Tyr Ser IleLys Asp Asn Ile Ser Asp Tyr Ile Asn Lys 995 1000 1005 TGG TTT TCC ATAACT ATT ACT AAT GAT AGA TTA GGT AAC GCA AAT ATT 3072 Trp Phe Ser Ile ThrIle Thr Asn Asp Arg Leu Gly Asn Ala Asn Ile 1010 1015 1020 TAT ATA AATGGA AGT TTG AAA AAA AGT GAA AAA ATT TTA AAC TTA GAT 3120 Tyr Ile Asn GlySer Leu Lys Lys Ser Glu Lys Ile Leu Asn Leu Asp 1025 1030 1035 1040 AGAATT AAT TCT AGT AAT GAT ATA GAC TTC AAA TTA ATT AAT TGT ACA 3168 Arg IleAsn Ser Ser Asn Asp Ile Asp Phe Lys Leu Ile Asn Cys Thr 1045 1050 1055GAT ACT ACT AAA TTT GTT TGG ATT AAG GAT TTT AAT ATT TTT GGT AGA 3216 AspThr Thr Lys Phe Val Trp Ile Lys Asp Phe Asn Ile Phe Gly Arg 1060 10651070 GAA TTA AAT GCT ACA GAA GTA TCT TCA CTA TAT TGG ATT CAA TCA TCT3264 Glu Leu Asn Ala Thr Glu Val Ser Ser Leu Tyr Trp Ile Gln Ser Ser1075 1080 1085 ACA AAT ACT TTA AAA GAT TTT TGG GGG AAT CCT TTA AGA TACGAT ACA 3312 Thr Asn Thr Leu Lys Asp Phe Trp Gly Asn Pro Leu Arg Tyr AspThr 1090 1095 1100 CAA TAC TAT CTG TTT AAT CAA GGT ATG CAA AAT ATC TATATA AAG TAT 3360 Gln Tyr Tyr Leu Phe Asn Gln Gly Met Gln Asn Ile Tyr IleLys Tyr 1105 1110 1115 1120 TTT AGT AAA GCT TCT ATG GGG GAA ACT GCA CCACGT ACA AAC TTT AAT 3408 Phe Ser Lys Ala Ser Met Gly Glu Thr Ala Pro ArgThr Asn Phe Asn 1125 1130 1135 AAT GCA GCA ATA AAT TAT CAA AAT TTA TATCTT GGT TTA CGA TTT ATT 3456 Asn Ala Ala Ile Asn Tyr Gln Asn Leu Tyr LeuGly Leu Arg Phe Ile 1140 1145 1150 ATA AAA AAA GCA TCA AAT TCT CGG AATATA AAT AAT GAT AAT ATA GTC 3504 Ile Lys Lys Ala Ser Asn Ser Arg Asn IleAsn Asn Asp Asn Ile Val 1155 1160 1165 AGA GAA GGA GAT TAT ATA TAT CTTAAT ATT GAT AAT ATT TCT GAT GAA 3552 Arg Glu Gly Asp Tyr Ile Tyr Leu AsnIle Asp Asn Ile Ser Asp Glu 1170 1175 1180 TCT TAC AGA GTA TAT GTT TTGGTG AAT TCT AAA GAA ATT CAA ACT CAA 3600 Ser Tyr Arg Val Tyr Val Leu ValAsn Ser Lys Glu Ile Gln Thr Gln 1185 1190 1195 1200 TTA TTT TTA GCA CCCATA AAT GAT GAT CCT ACG TTC TAT GAT GTA CTA 3648 Leu Phe Leu Ala Pro IleAsn Asp Asp Pro Thr Phe Tyr Asp Val Leu 1205 1210 1215 CAA ATA AAA AAATAT TAT GAA AAA ACA ACA TAT AAT TGT CAG ATA CTT 3696 Gln Ile Lys Lys TyrTyr Glu Lys Thr Thr Tyr Asn Cys Gln Ile Leu 1220 1225 1230 TGC GAA AAAGAT ACT AAA ACA TTT GGG CTG TTT GGA ATT GGT AAA TTT 3744 Cys Glu Lys AspThr Lys Thr Phe Gly Leu Phe Gly Ile Gly Lys Phe 1235 1240 1245 GTT AAAGAT TAT GGA TAT GTT TGG GAT ACC TAT GAT AAT TAT TTT TGC 3792 Val Lys AspTyr Gly Tyr Val Trp Asp Thr Tyr Asp Asn Tyr Phe Cys 1250 1255 1260 ATAAGT CAG TGG TAT CTC AGA AGA ATA TCT GAA AAT ATA AAT AAA TTA 3840 Ile SerGln Trp Tyr Leu Arg Arg Ile Ser Glu Asn Ile Asn Lys Leu 1265 1270 12751280 AGG TTG GGA TGT AAT TGG CAA TTC ATT CCC GTG GAT GAA GGA TGG ACA3888 Arg Leu Gly Cys Asn Trp Gln Phe Ile Pro Val Asp Glu Gly Trp Thr1285 1290 1295 GAA TAA 3894 Glu 1297 amino acids amino acid linearprotein 77 Met Pro Val Asn Ile Lys Asn Phe Asn Tyr Asn Asp Pro Ile AsnAsn 1 5 10 15 Asp Asp Ile Ile Met Met Glu Pro Phe Asn Asp Pro Gly ProGly Thr 20 25 30 Tyr Tyr Lys Ala Phe Arg Ile Ile Asp Arg Ile Trp Ile ValPro Glu 35 40 45 Arg Phe Thr Tyr Gly Phe Gln Pro Asp Gln Phe Asn Ala SerThr Gly 50 55 60 Val Phe Ser Lys Asp Val Tyr Glu Tyr Tyr Asp Pro Thr TyrLeu Lys 65 70 75 80 Thr Asp Ala Glu Lys Asp Lys Phe Leu Lys Thr Met IleLys Leu Phe 85 90 95 Asn Arg Ile Asn Ser Lys Pro Ser Gly Gln Arg Leu LeuAsp Met Ile 100 105 110 Val Asp Ala Ile Pro Tyr Leu Gly Asn Ala Ser ThrPro Pro Asp Lys 115 120 125 Phe Ala Ala Asn Val Ala Asn Val Ser Ile AsnLys Lys Ile Ile Gln 130 135 140 Pro Gly Ala Glu Asp Gln Ile Lys Gly LeuMet Thr Asn Leu Ile Ile 145 150 155 160 Phe Gly Pro Gly Pro Val Leu SerAsp Asn Phe Thr Asp Ser Met Ile 165 170 175 Met Asn Gly His Ser Pro IleSer Glu Gly Phe Gly Ala Arg Met Met 180 185 190 Ile Arg Phe Cys Pro SerCys Leu Asn Val Phe Asn Asn Val Gln Glu 195 200 205 Asn Lys Asp Thr SerIle Phe Ser Arg Arg Ala Tyr Phe Ala Asp Pro 210 215 220 Ala Leu Thr LeuMet His Glu Leu Ile His Val Leu His Gly Leu Tyr 225 230 235 240 Gly IleLys Ile Ser Asn Leu Pro Ile Thr Pro Asn Thr Lys Glu Phe 245 250 255 PheMet Gln His Ser Asp Pro Val Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270Gly Gly His Asp Pro Ser Val Ile Ser Pro Ser Thr Asp Met Asn Ile 275 280285 Tyr Asn Lys Ala Leu Gln Asn Phe Gln Asp Ile Ala Asn Arg Leu Asn 290295 300 Ile Val Ser Ser Ala Gln Gly Ser Gly Ile Asp Ile Ser Leu Tyr Lys305 310 315 320 Gln Ile Tyr Lys Asn Lys Tyr Asp Phe Val Glu Asp Pro AsnGly Lys 325 330 335 Tyr Ser Val Asp Lys Asp Lys Phe Asp Lys Leu Tyr LysAla Leu Met 340 345 350 Phe Gly Phe Thr Glu Thr Asn Leu Ala Gly Glu TyrGly Ile Lys Thr 355 360 365 Arg Tyr Ser Tyr Phe Ser Glu Tyr Leu Pro ProIle Lys Thr Glu Lys 370 375 380 Leu Leu Asp Asn Thr Ile Tyr Thr Gln AsnGlu Gly Phe Asn Ile Ala 385 390 395 400 Ser Lys Asn Leu Lys Thr Glu PheAsn Gly Gln Asn Lys Ala Val Asn 405 410 415 Lys Glu Ala Tyr Glu Glu IleSer Leu Glu His Leu Val Ile Tyr Arg 420 425 430 Ile Ala Met Cys Lys ProVal Met Tyr Lys Asn Thr Gly Lys Ser Glu 435 440 445 Gln Cys Ile Ile ValAsn Asn Glu Asp Leu Phe Phe Ile Ala Asn Lys 450 455 460 Asp Ser Phe SerLys Asp Leu Ala Lys Ala Glu Thr Ile Ala Tyr Asn 465 470 475 480 Thr GlnAsn Asn Thr Ile Glu Asn Asn Phe Ser Ile Asp Gln Leu Ile 485 490 495 LeuAsp Asn Asp Leu Ser Ser Gly Ile Asp Leu Pro Asn Glu Asn Thr 500 505 510Glu Pro Phe Thr Asn Phe Asp Asp Ile Asp Ile Pro Val Tyr Ile Lys 515 520525 Gln Ser Ala Leu Lys Lys Ile Phe Val Asp Gly Asp Ser Leu Phe Glu 530535 540 Tyr Leu His Ala Gln Thr Phe Pro Ser Asn Ile Glu Asn Leu Gln Leu545 550 555 560 Thr Asn Ser Leu Asn Asp Ala Leu Arg Asn Asn Asn Lys ValTyr Thr 565 570 575 Phe Phe Ser Thr Asn Leu Val Glu Lys Ala Asn Thr ValVal Gly Ala 580 585 590 Ser Leu Phe Val Asn Trp Val Lys Gly Val Ile AspAsp Phe Thr Ser 595 600 605 Glu Ser Thr Gln Lys Ser Thr Ile Asp Lys ValSer Asp Val Ser Ile 610 615 620 Ile Ile Pro Tyr Ile Gly Pro Ala Leu AsnVal Gly Asn Glu Thr Ala 625 630 635 640 Lys Glu Asn Phe Lys Asn Ala PheGlu Ile Gly Gly Ala Ala Ile Leu 645 650 655 Met Glu Phe Ile Pro Glu LeuIle Val Pro Ile Val Gly Phe Phe Thr 660 665 670 Leu Glu Ser Tyr Val GlyAsn Lys Gly His Ile Ile Met Thr Ile Ser 675 680 685 Asn Ala Leu Lys LysArg Asp Gln Lys Trp Thr Asp Met Tyr Gly Leu 690 695 700 Ile Val Ser GlnTrp Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile 705 710 715 720 Lys GluArg Met Tyr Asn Ala Leu Asn Asn Gln Ser Gln Ala Ile Glu 725 730 735 LysIle Ile Glu Asp Gln Tyr Asn Arg Tyr Ser Glu Glu Asp Lys Met 740 745 750Asn Ile Asn Ile Asp Phe Asn Asp Ile Asp Phe Lys Leu Asn Gln Ser 755 760765 Ile Asn Leu Ala Ile Asn Asn Ile Asp Asp Phe Ile Asn Gln Cys Ser 770775 780 Ile Ser Tyr Leu Met Asn Arg Met Ile Pro Leu Ala Val Lys Lys Leu785 790 795 800 Lys Asp Phe Asp Asp Asn Leu Lys Arg Asp Leu Leu Glu TyrIle Asp 805 810 815 Thr Asn Glu Leu Tyr Leu Leu Asp Glu Val Asn Ile LeuLys Ser Lys 820 825 830 Val Asn Arg His Leu Lys Asp Ser Ile Pro Phe AspLeu Ser Leu Tyr 835 840 845 Thr Lys Asp Thr Ile Leu Ile Gln Val Phe AsnAsn Tyr Ile Ser Asn 850 855 860 Ile Ser Ser Asn Ala Ile Leu Ser Leu SerTyr Arg Gly Gly Arg Leu 865 870 875 880 Ile Asp Ser Ser Gly Tyr Gly AlaThr Met Asn Val Gly Ser Asp Val 885 890 895 Ile Phe Asn Asp Ile Gly AsnGly Gln Phe Lys Leu Asn Asn Ser Glu 900 905 910 Asn Ser Asn Ile Thr AlaHis Gln Ser Lys Phe Val Val Tyr Asp Ser 915 920 925 Met Phe Asp Asn PheSer Ile Asn Phe Trp Val Arg Thr Pro Lys Tyr 930 935 940 Asn Asn Asn AspIle Gln Thr Tyr Leu Gln Asn Glu Tyr Thr Ile Ile 945 950 955 960 Ser CysIle Lys Asn Asp Ser Gly Trp Lys Val Ser Ile Lys Gly Asn 965 970 975 ArgIle Ile Trp Thr Leu Ile Asp Val Asn Ala Lys Ser Lys Ser Ile 980 985 990Phe Phe Glu Tyr Ser Ile Lys Asp Asn Ile Ser Asp Tyr Ile Asn Lys 995 10001005 Trp Phe Ser Ile Thr Ile Thr Asn Asp Arg Leu Gly Asn Ala Asn Ile1010 1015 1020 Tyr Ile Asn Gly Ser Leu Lys Lys Ser Glu Lys Ile Leu AsnLeu Asp 1025 1030 1035 1040 Arg Ile Asn Ser Ser Asn Asp Ile Asp Phe LysLeu Ile Asn Cys Thr 1045 1050 1055 Asp Thr Thr Lys Phe Val Trp Ile LysAsp Phe Asn Ile Phe Gly Arg 1060 1065 1070 Glu Leu Asn Ala Thr Glu ValSer Ser Leu Tyr Trp Ile Gln Ser Ser 1075 1080 1085 Thr Asn Thr Leu LysAsp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr 1090 1095 1100 Gln Tyr TyrLeu Phe Asn Gln Gly Met Gln Asn Ile Tyr Ile Lys Tyr 1105 1110 1115 1120Phe Ser Lys Ala Ser Met Gly Glu Thr Ala Pro Arg Thr Asn Phe Asn 11251130 1135 Asn Ala Ala Ile Asn Tyr Gln Asn Leu Tyr Leu Gly Leu Arg PheIle 1140 1145 1150 Ile Lys Lys Ala Ser Asn Ser Arg Asn Ile Asn Asn AspAsn Ile Val 1155 1160 1165 Arg Glu Gly Asp Tyr Ile Tyr Leu Asn Ile AspAsn Ile Ser Asp Glu 1170 1175 1180 Ser Tyr Arg Val Tyr Val Leu Val AsnSer Lys Glu Ile Gln Thr Gln 1185 1190 1195 1200 Leu Phe Leu Ala Pro IleAsn Asp Asp Pro Thr Phe Tyr Asp Val Leu 1205 1210 1215 Gln Ile Lys LysTyr Tyr Glu Lys Thr Thr Tyr Asn Cys Gln Ile Leu 1220 1225 1230 Cys GluLys Asp Thr Lys Thr Phe Gly Leu Phe Gly Ile Gly Lys Phe 1235 1240 1245Val Lys Asp Tyr Gly Tyr Val Trp Asp Thr Tyr Asp Asn Tyr Phe Cys 12501255 1260 Ile Ser Gln Trp Tyr Leu Arg Arg Ile Ser Glu Asn Ile Asn LysLeu 1265 1270 1275 1280 Arg Leu Gly Cys Asn Trp Gln Phe Ile Pro Val AspGlu Gly Trp Thr 1285 1290 1295 Glu 1535 base pairs nucleic acid doublelinear DNA (genomic) CDS 108..1526 78 AGATCTCGAT CCCGCGAAAT TAATACGACTCACTATAGGG GAATTGTGAG CGGATAACAA 60 TTCCCCTCTA GAAATAATTT TGTTTAACTTTAAGAAGGAG ATATACC ATG GGC CAT 116 Met Gly His 1 CAT CAT CAT CAT CAT CATCAT CAT CAC AGC AGC GGC CAT ATC GAA GGT 164 His His His His His His HisHis His Ser Ser Gly His Ile Glu Gly 5 10 15 CGT CAT ATG GCT AGC ATG GCTGAC ACA ATT TTA ATA CAA GTT TTT AAT 212 Arg His Met Ala Ser Met Ala AspThr Ile Leu Ile Gln Val Phe Asn 20 25 30 35 AAT TAT ATT AGT AAT ATT AGTAGT AAT GCT ATT TTA AGT TTA AGT TAT 260 Asn Tyr Ile Ser Asn Ile Ser SerAsn Ala Ile Leu Ser Leu Ser Tyr 40 45 50 AGA GGT GGG CGT TTA ATA GAT TCATCT GGA TAT GGT GCA ACT ATG AAT 308 Arg Gly Gly Arg Leu Ile Asp Ser SerGly Tyr Gly Ala Thr Met Asn 55 60 65 GTA GGT TCA GAT GTT ATC TTT AAT GATATA GGA AAT GGT CAA TTT AAA 356 Val Gly Ser Asp Val Ile Phe Asn Asp IleGly Asn Gly Gln Phe Lys 70 75 80 TTA AAT AAT TCT GAA AAT AGT AAT ATT ACGGCA CAT CAA AGT AAA TTC 404 Leu Asn Asn Ser Glu Asn Ser Asn Ile Thr AlaHis Gln Ser Lys Phe 85 90 95 GTT GTA TAT GAT AGT ATG TTT GAT AAT TTT AGCATT AAC TTT TGG GTA 452 Val Val Tyr Asp Ser Met Phe Asp Asn Phe Ser IleAsn Phe Trp Val 100 105 110 115 AGG ACT CCT AAA TAT AAT AAT AAT GAT ATACAA ACT TAT CTT CAA AAT 500 Arg Thr Pro Lys Tyr Asn Asn Asn Asp Ile GlnThr Tyr Leu Gln Asn 120 125 130 GAG TAT ACA ATA ATT AGT TGT ATA AAA AATGAC TCA GGA TGG AAA GTA 548 Glu Tyr Thr Ile Ile Ser Cys Ile Lys Asn AspSer Gly Trp Lys Val 135 140 145 TCT ATT AAG GGA AAT AGA ATA ATA TGG ACATTA ATA GAT GTT AAT GCA 596 Ser Ile Lys Gly Asn Arg Ile Ile Trp Thr LeuIle Asp Val Asn Ala 150 155 160 AAA TCT AAA TCA ATA TTT TTC GAA TAT AGTATA AAA GAT AAT ATA TCA 644 Lys Ser Lys Ser Ile Phe Phe Glu Tyr Ser IleLys Asp Asn Ile Ser 165 170 175 GAT TAT ATA AAT AAA TGG TTT TCC ATA ACTATT ACT AAT GAT AGA TTA 692 Asp Tyr Ile Asn Lys Trp Phe Ser Ile Thr IleThr Asn Asp Arg Leu 180 185 190 195 GGT AAC GCA AAT ATT TAT ATA AAT GGAAGT TTG AAA AAA AGT GAA AAA 740 Gly Asn Ala Asn Ile Tyr Ile Asn Gly SerLeu Lys Lys Ser Glu Lys 200 205 210 ATT TTA AAC TTA GAT AGA ATT AAT TCTAGT AAT GAT ATA GAC TTC AAA 788 Ile Leu Asn Leu Asp Arg Ile Asn Ser SerAsn Asp Ile Asp Phe Lys 215 220 225 TTA ATT AAT TGT ACA GAT ACT ACT AAATTT GTT TGG ATT AAG GAT TTT 836 Leu Ile Asn Cys Thr Asp Thr Thr Lys PheVal Trp Ile Lys Asp Phe 230 235 240 AAT ATT TTT GGT AGA GAA TTA AAT GCTACA GAA GTA TCT TCA CTA TAT 884 Asn Ile Phe Gly Arg Glu Leu Asn Ala ThrGlu Val Ser Ser Leu Tyr 245 250 255 TGG ATT CAA TCA TCT ACA AAT ACT TTAAAA GAT TTT TGG GGG AAT CCT 932 Trp Ile Gln Ser Ser Thr Asn Thr Leu LysAsp Phe Trp Gly Asn Pro 260 265 270 275 TTA AGA TAC GAT ACA CAA TAC TATCTG TTT AAT CAA GGT ATG CAA AAT 980 Leu Arg Tyr Asp Thr Gln Tyr Tyr LeuPhe Asn Gln Gly Met Gln Asn 280 285 290 ATC TAT ATA AAG TAT TTT AGT AAAGCT TCT ATG GGG GAA ACT GCA CCA 1028 Ile Tyr Ile Lys Tyr Phe Ser Lys AlaSer Met Gly Glu Thr Ala Pro 295 300 305 CGT ACA AAC TTT AAT AAT GCA GCAATA AAT TAT CAA AAT TTA TAT CTT 1076 Arg Thr Asn Phe Asn Asn Ala Ala IleAsn Tyr Gln Asn Leu Tyr Leu 310 315 320 GGT TTA CGA TTT ATT ATA AAA AAAGCA TCA AAT TCT CGG AAT ATA AAT 1124 Gly Leu Arg Phe Ile Ile Lys Lys AlaSer Asn Ser Arg Asn Ile Asn 325 330 335 AAT GAT AAT ATA GTC AGA GAA GGAGAT TAT ATA TAT CTT AAT ATT GAT 1172 Asn Asp Asn Ile Val Arg Glu Gly AspTyr Ile Tyr Leu Asn Ile Asp 340 345 350 355 AAT ATT TCT GAT GAA TCT TACAGA GTA TAT GTT TTG GTG AAT TCT AAA 1220 Asn Ile Ser Asp Glu Ser Tyr ArgVal Tyr Val Leu Val Asn Ser Lys 360 365 370 GAA ATT CAA ACT CAA TTA TTTTTA GCA CCC ATA AAT GAT GAT CCT ACG 1268 Glu Ile Gln Thr Gln Leu Phe LeuAla Pro Ile Asn Asp Asp Pro Thr 375 380 385 TTC TAT GAT GTA CTA CAA ATAAAA AAA TAT TAT GAA AAA ACA ACA TAT 1316 Phe Tyr Asp Val Leu Gln Ile LysLys Tyr Tyr Glu Lys Thr Thr Tyr 390 395 400 AAT TGT CAG ATA CTT TGC GAAAAA GAT ACT AAA ACA TTT GGG CTG TTT 1364 Asn Cys Gln Ile Leu Cys Glu LysAsp Thr Lys Thr Phe Gly Leu Phe 405 410 415 GGA ATT GGT AAA TTT GTT AAAGAT TAT GGA TAT GTT TGG GAT ACC TAT 1412 Gly Ile Gly Lys Phe Val Lys AspTyr Gly Tyr Val Trp Asp Thr Tyr 420 425 430 435 GAT AAT TAT TTT TGC ATAAGT CAG TGG TAT CTC AGA AGA ATA TCT GAA 1460 Asp Asn Tyr Phe Cys Ile SerGln Trp Tyr Leu Arg Arg Ile Ser Glu 440 445 450 AAT ATA AAT AAA TTA AGGTTG GGA TGT AAT TGG CAA TTC ATT CCC GTG 1508 Asn Ile Asn Lys Leu Arg LeuGly Cys Asn Trp Gln Phe Ile Pro Val 455 460 465 GAT GAA GGA TGG ACA GAATAACTCGAG 1535 Asp Glu Gly Trp Thr Glu 470 473 amino acids amino acidlinear protein 79 Met Gly His His His His His His His His His His SerSer Gly His 1 5 10 15 Ile Glu Gly Arg His Met Ala Ser Met Ala Asp ThrIle Leu Ile Gln 20 25 30 Val Phe Asn Asn Tyr Ile Ser Asn Ile Ser Ser AsnAla Ile Leu Ser 35 40 45 Leu Ser Tyr Arg Gly Gly Arg Leu Ile Asp Ser SerGly Tyr Gly Ala 50 55 60 Thr Met Asn Val Gly Ser Asp Val Ile Phe Asn AspIle Gly Asn Gly 65 70 75 80 Gln Phe Lys Leu Asn Asn Ser Glu Asn Ser AsnIle Thr Ala His Gln 85 90 95 Ser Lys Phe Val Val Tyr Asp Ser Met Phe AspAsn Phe Ser Ile Asn 100 105 110 Phe Trp Val Arg Thr Pro Lys Tyr Asn AsnAsn Asp Ile Gln Thr Tyr 115 120 125 Leu Gln Asn Glu Tyr Thr Ile Ile SerCys Ile Lys Asn Asp Ser Gly 130 135 140 Trp Lys Val Ser Ile Lys Gly AsnArg Ile Ile Trp Thr Leu Ile Asp 145 150 155 160 Val Asn Ala Lys Ser LysSer Ile Phe Phe Glu Tyr Ser Ile Lys Asp 165 170 175 Asn Ile Ser Asp TyrIle Asn Lys Trp Phe Ser Ile Thr Ile Thr Asn 180 185 190 Asp Arg Leu GlyAsn Ala Asn Ile Tyr Ile Asn Gly Ser Leu Lys Lys 195 200 205 Ser Glu LysIle Leu Asn Leu Asp Arg Ile Asn Ser Ser Asn Asp Ile 210 215 220 Asp PheLys Leu Ile Asn Cys Thr Asp Thr Thr Lys Phe Val Trp Ile 225 230 235 240Lys Asp Phe Asn Ile Phe Gly Arg Glu Leu Asn Ala Thr Glu Val Ser 245 250255 Ser Leu Tyr Trp Ile Gln Ser Ser Thr Asn Thr Leu Lys Asp Phe Trp 260265 270 Gly Asn Pro Leu Arg Tyr Asp Thr Gln Tyr Tyr Leu Phe Asn Gln Gly275 280 285 Met Gln Asn Ile Tyr Ile Lys Tyr Phe Ser Lys Ala Ser Met GlyGlu 290 295 300 Thr Ala Pro Arg Thr Asn Phe Asn Asn Ala Ala Ile Asn TyrGln Asn 305 310 315 320 Leu Tyr Leu Gly Leu Arg Phe Ile Ile Lys Lys AlaSer Asn Ser Arg 325 330 335 Asn Ile Asn Asn Asp Asn Ile Val Arg Glu GlyAsp Tyr Ile Tyr Leu 340 345 350 Asn Ile Asp Asn Ile Ser Asp Glu Ser TyrArg Val Tyr Val Leu Val 355 360 365 Asn Ser Lys Glu Ile Gln Thr Gln LeuPhe Leu Ala Pro Ile Asn Asp 370 375 380 Asp Pro Thr Phe Tyr Asp Val LeuGln Ile Lys Lys Tyr Tyr Glu Lys 385 390 395 400 Thr Thr Tyr Asn Cys GlnIle Leu Cys Glu Lys Asp Thr Lys Thr Phe 405 410 415 Gly Leu Phe Gly IleGly Lys Phe Val Lys Asp Tyr Gly Tyr Val Trp 420 425 430 Asp Thr Tyr AspAsn Tyr Phe Cys Ile Ser Gln Trp Tyr Leu Arg Arg 435 440 445 Ile Ser GluAsn Ile Asn Lys Leu Arg Leu Gly Cys Asn Trp Gln Phe 450 455 460 Ile ProVal Asp Glu Gly Trp Thr Glu 465 470 30 base pairs nucleic acid singlelinear other nucleic acid /desc = “DNA” 80 CGCCATGGCT GACACAATTTTAATACAAGT 30 32 base pairs nucleic acid single linear other nucleicacid /desc = “DNA” 81 GCCTCGAGTT ATTCTGTCCA TCCTTCATCC AC 32 12 aminoacids amino acid Not Relevant Not Relevant peptide Modified-site 12/note= “The asparagine residue at this position contains an amidegroup.” 82 Cys Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn 1 5 10

1. A host cell containing a recombinant expression vector, said vectorencoding a protein comprising at least a portion of a Clostridiumbotulinum toxin, said toxin selected from the group consisting of type Btoxin and type E toxin.
 2. The host cell of claim 1, wherein and saidhost cell is capable of expressing said protein at a level greater thanor equal to 5% of the total cellular protein.
 3. The host cell of claim1, wherein and said host cell is capable of expressing said protein as asoluble protein at a level greater than or equal to 0.25% of the totalsoluble cellular protein.
 4. The host cell of claim 1, wherein said hostcell is an Escherichia coli cell.
 5. The host cell of claim 1, whereinsaid host cell is an insect cell.
 6. The host cell of claim 1, whereinsaid host cell is a yeast cell.
 7. A host cell containing a recombinantexpression vector, said vector encoding a fusion protein comprising anon-toxin protein sequence and at least a portion of a Clostridiumbotulinum toxin, said toxin selected from the group consisting of type Btoxin and type E toxin.
 8. The host cell of claim 7, wherein saidportion of said toxin comprises the receptor binding domain.
 9. The hostcell of claim 7, wherein said non-toxin protein sequence comprises apoly-histidine tract.
 10. A vaccine comprising a fusion protein, saidfusion protein comprising a non-toxin protein sequence and at least aportion of a Clostridium botulinum toxin, said toxin selected from thegroup consisting of type B toxin and type E toxin.
 11. The vaccine ofclaim 10 further comprising a fusion protein comprising a non-toxinprotein sequence and at least a portion of Clostridium botulinum type Atoxin.
 12. The vaccine of claim 10, wherein said portion of saidClostridium botulinum toxin comprises the receptor binding domain. 13.The vaccine of claim 10 wherein said non-toxin protein sequencecomprises a poly-histidine tract.
 14. The vaccine of claim 10, whereinsaid vaccine is substantially endotoxin-free.
 15. A method of generatingantibody directed against a Clostridium botulinum toxin comprising: a)providing in any order: i) an antigen comprising a fusion proteincomprising a non-toxin protein sequence and at least a portion of aClostridium botulinum toxin, said toxin selected from the groupconsisting of type B toxin and type E toxin, and ii) a host; and b)immunizing said host with said antigen so as to generate an antibody.16. The method of claim 15, wherein said antigen further comprises afusion protein comprising a non-toxin protein sequence and at least aportion of Clostridium botulinum type A toxin.
 17. The method of claim15, wherein said portion of said Clostridium botulinum toxin comprisesthe receptor binding domain.
 18. The method of claim 15 wherein saidnon-toxin protein sequence comprises a poly-histidine tract.
 19. Themethod of claim 15 wherein said host is a mammal.
 20. The method ofclaim 19 wherein said mammal is a human.
 21. The method of claim 15further comprising step c) collecting said antibodies from said host.22. The method of claim 21 further comprising step d) purifying saidantibodies.
 23. The antibody raised according to the method of claim 15.24. The antibody raised according to the method of claim 16.